Revue Méd. Vét., 2019, 170, 1-3, 15-21

EFFECTS OF TARANTULA CUBENSIS AND NERIUM OLEANDER ON CANCER 15

Introduction

Colorectal cancer is one of the most common types of cancer in human medicine. Colorectal cancer is responsible for 10% of deaths in cancer cases. Genetic factors and especially diet play the main roles in the aetiology [13, 45]. Colorectal cancer may also be observed in pet clinics [3, 44]. Apart from classical therapy, alternative medicine is being investigated in the treatment of the disease [8].

In colon cancer, abnormal crypts are detected in the colon mucosa and defined as aberrant cryptal foci (ACF), which is accepted as a biomarker [16, 40]. It is known that chronic inflammation may lead to cancer [29], where immune system cells secret cytokines, which can be proinflammatory or

anti-inflammatory, according to their functions in the body. While tumour necrosis factor (TNF)-α and interleukin-6 (IL-6) are defined as proinflammatory cytokines, IL-10 is defined as an anti-inflammatory cytokine [39]. Increased proinflammatory cytokine levels are reported in colorectal cancers [13, 29], whereas IL-10 inhibits tumour growth by modulating apoptosis and suppressing angiogenesis [28]. Secreted IL-2 from activated T helper cells shows anticancer effect [2, 19].

The cyclooxygenase (COX)-2 enzyme, which is an inducible COX species, is involved in stimulating the synthesis of prostaglandins (PGs) during cancer progression. PGE2, the final product produced by COX-2, has a carcinogenic effect by stimulating angiogenesis and inhibiting apoptosis

SUMMARY

The aim of this research was to determine the effects of Tarantula cubensis alcoholic extract (TCAE) and Nerium oleander distillation (NOD) on the colon aberrant cryptal foci (ACF) score, serum cytokines [Tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-6, IL-10], prostaglandin E2, and oxidative status parameters (malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase) in an experimentally induced colon cancer model in rats. Male rats (38 in total) were divided into 4 groups: Control group (n = 8), Colon Cancer group (CC, n = 10), CC+TCAE group (n = 10), and CC+NOD group (n = 10). Except for the Control group, the rest of rats received azoxymethane (15 mg/kg, SC) once a week for two weeks to induce colon cancer. TCAE was administered once a week at 0.2 mL/kg (SC) and NOD was administered in water to CC+TCAE and CC+NOD groups for 18 weeks. After blood samples were collected, the rats were euthanized, colons were removed, washed with saline, then fixed with 10% formaldehyde, and stained with 0.2% methylene blue. ACFs were scored by light microscopy. Serum levels of cytokines, prostaglandin E2, and oxidative status parameters were determined with ELISA. ACF score of the CC group was higher than CC+TCAE and CC+NOD groups. The prostaglandin E2 level of the Control group was lower than the CC+TCAE group, while IL-2 and IL-10 levels of the Control group were lower than all other groups. In conclusion, it can be stated that TCAE and NOD applications may be beneficial in the treatment of colon cancer.

Keywords: Tarantula cubensis alcoholic extract, Nerium oleander distillate, colon cancer

RÉSUMÉ

Effets de l’extrait alcoolique de Tarantula cubensis et du distillat de Nerium oleander sur le cancer du côlon expérimentalement induit chez le rat

Le but de cette étude était de déterminer les effets de l’extrait alcoolique de Tarantula cubensis (TCAE) et du distillat de Nerium oleander (NOD) sur les scores de formation de cryptes coliques aberrantes focales (ACF), les cytokines sériques [facteur de nécrose tumorale (TNF)-α, interleukine (IL)-2, IL-6, IL-10], prostaglandine E2, et des paramètres de l’état oxydatif (malondialdéhyde, superoxyde dismutase, catalase, glutathion peroxydase) dans un cancer du côlon induit expérimentalement chez le rat. Des rats mâles (38 au total) ont été répartis en 4 groupes : contrôle (n = 8), groupe cancer du côlon (CC, n = 10), groupe CC+TCAE (n = 10), et le groupe CC+NOD (n = 10). À l’exception du groupe témoin, le reste des rats a reçu de l’azoxyméthane (15 mg/kg, SC) une fois par semaine pendant deux semaines pour induire le cancer du côlon. Le TCAE a été administré une fois par semaine à raison de 0,2 ml/kg (SC) et le La NOD a été administrée dans l’eau aux groupes CC+TCAE et CC+NOD pendant 18 semaines. Après le prélèvement d’échantillons de sang, les rats ont été euthanasiés, les colons ont été enlevés, lavés avec une solution saline, puis fixés avec 10 % de formaldéhyde, et teinté avec 0,2% de bleu de méthylène. Les ACF ont été notés par microscopie optique. Les taux sériques de cytokines, prostaglandine E2 et les paramètres de l’état oxydatif ont été déterminées avec ELISA. Le score ACF du groupe CC était plus élevé que celui des groupes CC+TCAE et CC+NOD. Le niveau de prostaglandine E2 du contrôle était inférieur à celui du groupe CC+TCAE, tandis que les taux d’IL-2 et d’IL-10 étaient inférieurs à ceux du groupe CC+TCAE. En conclusion, l’administration des extraits TCAE et NOD peut être bénéfiques dans le traitement du cancer du côlon expérimentalement induit chez le rat.

Mots-clefs : cancer, colon, rat, extraits plantes

Effects of Tarantula cubensis alcoholic extract and Nerium oleander distillate on experimentally induced colon cancerA. ER1*, O. OZDEMIR2, D. COSKUN3, B. DIK1, E. BAHCIVAN4, H. ESER FAKI5, E. YAZAR1

1Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Selcuk University, Campus, Selcuklu, 42075, Konya, Turkey2Department of Pathology, Faculty of Veterinary Medicine, Selcuk University, Campus, Selcuklu, 42075, Konya, Turkey3Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Siirt University, 56100, Siirt, Turkey4Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Kafkas University, 36000, Kars, Turkey5Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Dicle University, 21000, Diyarbakir, Turkey

Corresponding author: aer@selcuk.edu.tr

Revue Méd. Vét., 2019, 170, 1-3, 15-21

ER (A.) AND COLLABORATORS16

in tumour cells [18]. It has been reported that COX inhibitors can be used in the treatment of canine mammary cancer [22] and COX levels are higher in the experimental colon cancer group than the control group [35].

Reactive oxygen species (ROS, singlet oxygen, superoxide radical, hydroxyl radical, hydrogen peroxide, hypochloric acid, peroxynitrite, etc.) play a role in many diseases, including cancer. Cells protect themselves against the harmful effects of ROS through enzymatic antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT), and nonenzymatic substances. When ROS cannot be sufficiently detoxified, lipid peroxidation (known as oxidative stress) develops in the cells. Lipid peroxidation can cause damage to cells that can lead to cell death. Malondialdehyde (MDA) occurs as one of the end-products resulting from lipid peroxidation and MDA measurement is most useful in determining oxidative stress in biological materials. Research has shown that antioxidant substances can prevent cancer development [45, 47].

Tarantula cubensis alcoholic extract (TCAE) is marketed as a homeopathic product in veterinary medicine. The manufacturer reports that TCAE stimulates the demarcation of necrotic tissue, accelerates epithelisation and wound healing, shows anti-inflammatory effect, and stimulates resorption to relieve oedema and swelling. Considering these effects, it has been suggested that TCAE can be used in traumatic and infectious diseases of the skin, foot, udder, and birth canal [36]. In addition, an anticancer effect has been reported for TCAE in canine mammary cancer [22].

Nerium oleander (NO, oleander) is grown as an ornamental plant in the Mediterranean region. As all parts of NO are toxic, their use for medical purposes is limited. The oleandrin contained in the plant causes severe cardiotoxicity [5]. Although use of NO in medicine is limited, it may have anticancer activity [6] and an extract of NO may show beneficial effect in pancreatic cancer [33]. While extracts of the plant may contain toxic compounds, it has been reported that NO distillate (NOD) has no toxic effect when administered orally to rats in the acute oral toxicity test according to the recommendations of the World Economic Development and Cooperation Organization (OECD). After 14 days, it shows no unwanted effects on a hemogram, in routine biochemical parameters, or macroscopic and microscopic pathological lesions [10]. In addition, it has been reported that NOD has no teratogenic effect [21] and has beneficial effects in Type II diabetes and cholesterol metabolism after oral application [4, 23, 48].

Considering the anticancer effects of TCAE [12, 22] and NO [6, 33] in other types of cancer, it is hypothesized that they may also have positive effects in experimental colon cancer.

The aim of the current research was to determine the effects of TCAE and NOD treatments in an experimentally

induced colon cancer model in rats, as reported through the ACF scores, the levels of serum cytokines (TNFα, IL-2, IL-6, IL-10), prostaglandin E2, and oxidative status parameters (MDA, SOD, GPX, CAT).

Material and Methods

Wistar Albino male rats (38 in total, 12-16 weeks, 242 ± 1.92 g) were used in this study, utilizing research procedures approved by the ethics committee (SUDAM, 2016-45). During the experimental period, the rats were housed in standard rat cages in a central facility under controlled conditions (12 h light/dark cycle and room temperature of 20°C ± 2°C) at SUDAM and allowed water and food ad libitum. NOD was obtained using a previously described method [4]. In this study, rats were divided into 4 groups with a Control group (C) consisting of 8 rats and the remaining 30 rats divided into 3 equal groups. The Colon Cancer group (CC, n = 10) received azoxymethane (15 mg/kg, SC) once a week for two weeks to induce colon cancer [35]. The CC+TCAE group (n = 10) received azoxymethane (15 mg/kg, SC) once a week for two weeks to induce colon cancer and TCAE administered subcutaneously once a week at the dose of 0.2 mL/kg during the experimental period. The CC+NOD group (n = 10) received azoxymethane (15 mg/kg, SC) once a week for two weeks to induce colon cancer and NOD was drunk for 18 weeks.

At the end of the experiment, blood samples were taken from the heart and the rats were euthanized by cervical dislocation under anaesthesia (ketamine 95 mg/kg, SC + xylazine 5 mg/kg, SC). Immediately after the euthanasia, the colons were removed, elongated, and washed with saline, then fixed with 10% formalin and stained with 0.2% methylene blue. ACFs were scored according to a previously reported method [25] and assessed by light microscopy (Olympus Corporation, Tokyo, Japan). The ACF scores were determined using 15 randomly selected crypts in each rat colon and scored as follows: score 1: 1-3 crypts, score 2: 4-6 crypts, score 3: 7-9 crypts and score 4: >10 crypts. In addition, macroscopically observed tumour mass scores were assessed as follows: score 1: 1 mass between 0.1 and 0.2 cm; Score 2: 2 masses between 0.1 and 0.2 cm or 1 mass between 0.3 and 0.5 cm; Score 3: 2 or 3 masses between 0.3 and 0.5 cm or 1 mass between 0.5 and 1 cm; Score 4: 2 or 3 masses between 0.5 and 1 cm or 1 mass between 1 and 1.5 cm; and Score 5: 1 mass > 1.5 cm. In addition, a histopathological examination of the large intestine was performed using a routine staining technique.

Serum TNFα (Rat TNFα ELISA kit, Elabscience, Texas, USA), IL-2 (Rat IL-2 ELISA kit, Elabscience, Texas, USA), IL-6 (Rat IL-6 ELISA kit, Elabscience, Texas, USA), IL-10 (Rat IL-10 ELISA kit, Elabscience, Texas, USA), prostaglandin E2 (PGE2 Elabscience, Texas, USA), MDA (MDA ELISA kit, Elabscience, Texas, USA), SOD (SOD ELISA kit, Elabscience, Texas, USA), CAT (CAT ELISA kit, SunRed Biotechnology Company, Shangai, China) and

Revue Méd. Vét., 2019, 170, 1-3, 15-21

EFFECTS OF TARANTULA CUBENSIS AND NERIUM OLEANDER ON CANCER 17

GPX (GPX ELISA kit, Elabscience, Texas, USA) levels were determined with commercially available kits using an ELISA reader (MWGt Lambda Scan 200, Bio-Tek Instruments Inc., VT, USA) according to manufacturer assaying protocols.

Statistically analyses were performed from 7 animals obtained from each group. ACF scores were evaluated using a Mann-Whitney U test, while the all other data was evaluated by ANOVA and Tukey post hoc tests (SPSS 22.0). ACF scores were presented as median, whereas other parameters were shown as mean ± standard error (SE). P<0.05 levels were accepted as statistically significant.

Results

ACF scores (Figure 1), serum cytokines levels, (TNFα, IL-2, IL-6, IL-10), PGE2 levels, and oxidative status parameters (MDA, SOD, CAT, GPX) are shown in Table I. ACF scores for the CC group were higher (P<0.05) than the CC+TCAE and CC+NOD groups, while ACF scores for the CC+TCAE and CC+NOD groups were similar (P>0.05). In histopathological examinations, ACF were detected in the experimental groups (Figure 3). These structures were dyed darker than normal crypt epithelium and presented different lumen shapes and organizations. ACFs were more prominent in the sections parallel to the mucosa. In macroscopic examinations, nodular tumours were seen located in the mucosal surface as sessile (Figure 4). In these areas, it was observed that the tumoral crypts spread to the submucosa and there was an increase in connective tissue around the crypts with inflammatory reactions. In addition, there were a large number of sessile-like nodular structures ranging from 2 to 5 mm in diameter in three animals in the CC group. In the CC+NOD group, tumoral structures with a diameter of 1 to 5 mm were observed in three animals, while two animals in the CC+TCAE group had tumoral structures with a diameter of

1 to 2 mm (Figure 2). The tumoral structures in the treatment groups were smaller than those found in the cancer group. Total macroscopic tumour mass scores were 7, 2 and 4 in the CC, CC+TCAE and CC+NOD groups, respectively (Table II). No pathology was observed in the C group.

Parameters Control (n:7)

CC (n:7)

CC+TCAE (n:7)

CC+NOD (n:7)

ACF 0c 3a 2b 2b

BW begin (g) 241.42±3.69 242.00±3.92 242.57±4.11 243.42±4.50BW finish (g) 538.85±15.69 481.85±14.85 506.14±25.08 524.14±15.79TNFα pg/mL 124.28±97.01 297.85±159.02 708.57±297.61 376.66±159.52IL-2 pg/mL 270.85±104.60b 1404.00±151.93a 988.28±177.03a 1024.85±210.68a

IL-6 pg/mL 317.82±68.41 329.40±134.64 329.26±98.88 230.21±121.97IL-10 pg/mL 15.55±9.38c 305.66±14.98a 311.62±5.56a 173.91±59.57b

PGE2 pg/mL 313.20±58.86b 512.25±53.86ab 560.03±54.56a 535.11±61.49ab

MDA ng/mL 183.72±17.74 266.65±34.87 226.51±18.96 277.43±22.46SOD ng/mL 2.61±0.07 2.64±0.07 2.86±0.03 2.67±0.11CAT ng/mL 24.51±6.25 20.30±1.31 15.99±2.03 15.53±1.18GPX ng/dL 18.72±0.69 16.21±1.26 16.57±1.76 13.62±1.65

BW: Body weight, ACF: Aberrant cryptal foci, TNFα: Tumour necrosis factor alpha, IL: Interleukin, PGE2: Prostaglandin E2, MDA: Malondialdehyde, SOD: Superoxide dismutase, CAT: Catalase, GPX: Glutathione peroxidase. a, b, c: The different letters in the same line are statistically significant (P<0.05).

Table I: Effects of TCAE and NOD administration on the ACF scores, levels of serum cytokines, levels of prostaglandin E2, and oxidative status parameters (mean±SE)

Figure 1: Aberrant cryptal foci (arrows) stained with methylene blue

Revue Méd. Vét., 2019, 170, 1-3, 15-21

ER (A.) AND COLLABORATORS18

IL-2 and IL-10 levels for the Control group were lower (P<0.05) than all cancer groups, whereas the PGE2 level of the CC+TCAE group was higher (P<0.05) than the C group.

Discussion

Although colorectal cancer is one of the common types of cancer in humans [45], it may be also observed in dogs and pets in the veterinary clinic [3, 42, 44, 49]. In addition to classical methods, alternative methods of treatment have been explored [8].

In this study, the ACF score (a diagnostic biomarker of colorectal cancer [16, 34, 40]) observed for the CC group was higher than those obtained for the CC+TCAE and CC+NOD groups (P<0.05, Figure 1), where the Control group showed no aberrant cryptal foci. In the macroscopic examinations, TCAE administration reduced the tumour mass score by 73% and NOD application by 43% compared to the CC group (Table II, Figure 2). No tumour mass was observed in the Control group. In the histopathological examinations, ACFs formed in the experimental groups were darker than normal crypt epithelium and presented different lumen shapes and organizations (Figure 3). In macroscopic examinations, developed sessile adenomas were observed in the nodules on the surface of the mucosa, tumoral crypts were spread to the submucosa, and inflammation reactions were observed around these crypts (Figure 4). Similar pathological findings have been reported in experimental studies with azoxymethane [8, 31, 35]. In the current study, it was determined that TCAE administration decreased the ACF score level compared to CC group (P<0.05, Table I, Figure 1). It has been reported that TCAE may accelerate

Figure 2: Macroscopic nodular structures (arrows) in cancer groups

Figure 3: Aberrant cryptal foci of colon stained with H&E

Figure 4: Sessile tumour masses (red arrows), and histopathological appearances of tumoral crypts (black arrows), connective tissue (ct) and inflammatory reactions (ir) in the colon of the cancer groups, H&E.

Revue Méd. Vét., 2019, 170, 1-3, 15-21

EFFECTS OF TARANTULA CUBENSIS AND NERIUM OLEANDER ON CANCER 19

epithelisation [37] and may have beneficial effects on wound healing [1], ulcer [46] and papillomatosis [17, 32]. In addition, it has been reported that TCAE can show anticancer activity in a cancer cell line [14] and can control cell growth by inducing apoptosis in canine mammary cancer cells [15, 22]. In this study, it was determined that NOD application to CC group animals decreased ACF score levels compared to the CC group (P<0.05, Table I). NO may show beneficial effects in pancreatic cancer [33] as anticancer activities have been observed in plant-based investigations of its antioxidant properties [31, 38, 43]. When considering the role of ROS in cancer formation [45], the anticancer activities of TCAE and NOD can be attributed to the antioxidant effect of both agents [9, 11, 20].

Increases in the TNFα levels of the CC+TCAE group were five times higher than the Control group, although there was no statistically significant change in the levels of TNFα and IL-6 (P>0.05, Table I). It has been reported that high TNFα levels are observed in azoxymethane-induced experimental colon cancer [8], whereas non-statistical fluctuations were observed in TNFα and IL-6 levels after TCAE administration in healthy sheep [7]. In cancer, TNFα can have different effects, with low levels of TNFα being carcinogenic by inhibiting DNA repair damage and stimulating angiogenic factors, while high levels of TNFα have anticancer effects by destroying the vessels and necrotic effects in tumours. There is also a positive correlation between the size of some tumours and TNF levels [13]. The anticancer effect of TCAE may be attributed to its stimulating effect on TNF-α.

In the current study, the levels of IL-2 which has anticancer effect [24] were significantly higher in the cancer groups than those observed for the Control group (P<0.05, Table I). IL-2 is secreted from activated T helper cells, activates macrophages and lymphocytes, stimulates lymphokine release, and causes lysis of cancer cells [19, 24]. IL-2 has been approved by the Food and Drug Administration (FDA) for use in metastatic melanoma and renal cell carcinoma [27]. Increased IL-2 levels in the cancer group may be immunological responses to the developing cancer, where TCAE and NOD have no any effect on IL-2 synthesis.

IL-10 levels in the cancer groups were also higher than the Control group (P<0.05, Table I). It has been reported that TCAE caused non-statistical fluctuations in the IL-10 levels in sheep [7]. Considering that IL-10 has antitumor effects, acting as an inhibitor of both proinflammatory cytokine synthesis and angiogenesis [26, 28], the high levels of IL-10 observed in the cancer groups of the present study were found to be normal immunological responses to inflammation and cancer assessable. It may also suggest that NOD partially inhibits IL-10 synthesis, whereas TCAE administration does not have an inhibitory effect on IL-10 synthesis.

In the present study, the PGE2 levels of the cancer groups were generally higher than the Control group, while levels for the CC+TCAE were statistically different (P<0.05, Table I). Relations between inflammation and colorectal cancer have been reported [13], as have increased COX and PGE2 levels in colon and colorectal cancer and the tumorigenic effect of PGE2 [30, 35, 41]. In addition, increased COX level in cancer may stimulate cell proliferation, inhibit apoptosis, and play a role in angiogenesis and metastasis [22, 29]. Hence, it has been reported that the use of COX inhibitors, which inhibit prostaglandin synthesis, may reduce the risk of cancer [29, 41]. In this study, elevated PGE2 levels in the cancer groups may be derived from stimulation of COX enzyme in the colon. In addition, the results suggest that NOD has more anti-inflammatory effect than TCAE when considering the measured PGE2 levels.

In this study, no changes were observed in the MDA levels (a primary biomarker of oxidative stress) or the antioxidant enzymes (P> 0.05, Table I). Oxygen and nitrogen-derived free radicals produced in the cells can cause cancer by creating oxidative stress [26, 45]. The produced ROS is inactivated by antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) in the cell and it is known that antioxidant substances may exhibit anticancer activity [45, 47]. Although antioxidant activities for NOD [11] and TCAE [9, 20] were reported, changes in the serum oxidative status parameters were not observed in the current study. These results may be derived from the induced cancer model, animal kinds, and dosing regimens of TCAE and NOD.

Rats Control CC CC+TCAE CC+NOD1 0 0 0 22 0 0 0 03 0 0 0 04 0 2 0 05 0 2 0 06 0 0 1 17 0 3 1 1Total score 0 7 2 4

Table II: Distribution of macroscopic tumoral nodules and scores in the colon

Revue Méd. Vét., 2019, 170, 1-3, 15-21

ER (A.) AND COLLABORATORS20

Conclusions

In conclusion, TCAE and NOD may show anticancer properties in colon cancer, and these properties may be derived from their stimulating effect on cytokines. In addition, TCAE may show more anticancer effect than NOD via stimulating the synthesis of TNF-α.

Acknowledgements

This study was financed by SUBAPK (17401073). Study abstract was present as oral presentation in the International Conference on Veterinary, Agriculture and Life Sciences (ICVALS), October 26-29, 2018, Antalya, Turkey

Conflict of interests

The authors declare that there is no conflict of interest regarding the publication of this paper.

References

1. - ADIB-HASHEMI F., FARAHMAND F., HESARI S.F., REZAKHANIHA B., FALLAH E., FAYYAZ A.F., DADPAY M. : Anti-inflammatory and protective investigations on the effects of Theranekron® “an alcoholic extract of the Tarantula cubensis” on wound healing of peritoneal in the rat: an in vivo comparative study. Diag. Pathol., 2015, 10, 19.

2. - ARENAS-RAMIREZ N., WOYTSCHAK J., BOYMAN O. : Interleukin-2: Biology, design and application. Trends Immunol., 2015, 36, 763-777.

3. - ARTEAGA T.A., MCKNIGHT J., BERGMAN P.J. : A review of 18 cases of feline colonic adenocarcinoma treated with subtotal colectomies and adjuvant carboplatin. J. Am. Anim. Hosp. Assoc., 2012, 48, 399-404.

4. - BAS A.L., DEMIRCI S., YAZIHAN N., UNEY K., ERMIS KAYA E. : Nerium oleander distillate improves fat and glucose metabolism in high-fat diet-fed streptozotocin-induced diabetic rats. Int. J. Endocrinol., 2012, doi: 10.1155/2012/947187.

5. - BAYTOP T. : Herbal Medicine in Turkey. Istanbul University Press, Istanbul, Turkey, 1984, 411p.

6. - CALDERON-MONTANO J.M., BURGOS-MORON E., ORTA M.L., MATEOS S., LOPEZ-LAZARO M. : A hydroalcoholic extract from the leaves of Nerium oleander inhibits glycolysis and induces selective killing of lung cancer cells. Planta Med., 2013, 79, 1017-1023.

7. - CORUM O., ER A., DIK B. : Investigation of the effect of Tarantula cubensis extract on acute phase response. Acta Sci. Vet., 2016, 44, 1414.

8. - CORUM O., OZDEMIR O., YAZAR E. : Halofuginone may suppresses azoxymethane-induced cytokines serum tumor necrosis factor-α synthesis and aberrant crypt foci progression in rat colon. Indian J. Anim. Res., 2017, 51, 1120-1124.

9. - DIK B., ER A., CORUM O. : Effect of alcoholic extract of Tarantula cubensis (Theranekron®) on serum thiobarbituric acid-reactive species concentrations in sheep. Eurasian J. Vet. Sci., 2014, 30, 68-71.

10. - DIK B., UNEY K., OZDEMIR O., DEMIRCI S., YAZIHAN N.A., BAS A.L. : Acute oral toxicity of Nerium oleander distillate in rats. J. Vet. Pharmacol. Therap., 2012, 35, 78-102.

11. - ER A., CORUM O., CETIN G., DIK B. : Effect of Nerium oleander distillate administration on serum nitric oxide level. Eurasian J. Vet. Sci., 2015, 31, 70-74.

12. - ER A., CORUM O., CORUM D., HITIT M., DONMEZ H., GUZELOGLU A. : Alcoholic extract of Tarantula cubensis induces apoptosis in MCF-7 cell line. Biomed Res., 2017, 28, 3660-3665.

13. - ERARSLAN E., YUKSEL I., HAZNEDAROGLU S.  : The relation between colorectal carcinogenesis and metabolic syndrome. Cumhuriyet Med. J., 2012, 34, 380-385.

14. - GHASEMI-DIZGAH A., NAMI B., AMIRMOZAFARI N. : Tarantula cubensis venom (Theranekron®) selectively destroys human cancer cells via activating caspase-3-mediated apoptosis. Acta Med. Int., 2017, 4, 73-79.

15. - GULTIKEN N., GUVENC T., KAYA D., AGAOGLU A.R., AY S.S., KUCUKASLAN I., EMRE B., FINDIK M., SCHAFER-SOMI S., ASLAN S. : Tarantula cubensis extract alters the degree of apoptosis and mitosis in canine mammary adenocarcinomas. J. Vet. Sci., 2015, 16, 213-219.

16. - GUPTA A.K., SCHOEN R.E.: Aberrant crypt foci: are they intermediate endpoints of colon carcinogenesis in humans? Curr. Opin. Gastroenterol., 2009, 25, 59-65.

17. - ICEN H., SEKIN S., SIMSEK A., KOCHAN A., TUNIK S. : The efficacy of Tarantula cubensis extract (Theranekron) in treatment of canine oral papillomatosis. Asian J. Anim. Vet. Adv., 2011, 6, 744-749.

18. - JANAKIRAM N.B., RAO C.V. : The role of inflammation in colon cancer. In: B.B. AGGARWAL, B. GUPTA (Eds): Inflammation and Cancer, Springer, NY, USA, 2014, pp. 25-52.

19. - KAPUCU S. : Nursing management and interleukin-2 use in patients with cancer. J. Hacettepe Uni. Fac. Nursing, 2015, 2, 49-59.

20. - KARABACAK M., ERASLAN G., KANBUR M., SARICA Z.S. : Effects of Tarantula cubensis D6 on aflatoxin-induced injury in biochemical parameters in rats. Homeopathy, 2015, 104, 205-210.

21. - KARABULUT A.K., UYSAL I.I., BAS A.L., DOGAN N.U., FAZLIOGULLARI Z., ACAR H. : Investigation of developmental toxicity and teratogenicity of lyophylised aqueous distillate of Nerium oleander on rat embryos cultured in vitro. Genel Tip. Derg., 2014, 24, 58-63.

22. - KARAYANNOPOULOU M., LAFIONIATIS S. : Recent advances on canine mammary cancer chemotherapy:

Revue Méd. Vét., 2019, 170, 1-3, 15-21

EFFECTS OF TARANTULA CUBENSIS AND NERIUM OLEANDER ON CANCER 21

A review of studies from 2000 to date. Revue Med. Vet., 2016, 167, 7-8.

23. - KARS M.D., ODABASI B.A., KARS G., UNEY K., BAGCI Y., BAS A.L. : Implications from a pharmacogenomic analysis: Nerium oleander leaf distillate supplemented diet regulates cholesterol metabolism in rats. Pharm. Biol., 2014, 52, 988-993.

24. - KELLE I. : Biotoxins in cancer therapy. Dicle Tıp Der., 2007, 34, 226-232.

25. - KRISTIANSEN E. : The role of aberrant crypt foci induced by the two heterocyclic amines 2-amino-3-methyl-imidazo[4,5jJquinoline (IQ) and 2-amino- 1 -methyl-6-phenyl-imidazo[4,5b]pyridine (PhIP) in the development of colon cancer in mice. Cancer Lett., 1996, 110, 187-192.

26. - LANDSKRON G., de la FUENTE M., THUWAJIT P., THUWAJIT C., HERMOSO M.A. : Chronic inflammation and cytokines in the tumor microenvironment. J. Immunol. Res., 2014, 149185.

27. - LEE S., MARGOLIN K. : Cytokines in cancer immunotherapy. Cancers, 2011, 3, 3856-3893.

28. - LIN W.W., KARIN M. : A cytokine-mediated link between innate immunity, inflammation, and cancer. J. Clin. Invest., 2007, 117, 1175-1183.

29. - LU H., OUYANG W., HUANG C. : Inflammation, a key event in cancer development. Mol. Cancer Res., 2006, 4, 221-233.

30. - MOREIRA L., CASTELLS A. : Cyclooxygenase as a target for colorectal cancer chemoprevention. Curr. Drug Targets., 2011, 12, 1888-1894.

31. - NASIR N.L., KAMSANI N.E., MOHTARRUDIN N., OTHMAN F., TOHID S.F., ZAKARIA Z.A. : Anticarcinogenic activity of Muntingia calabura leaves methanol extract against the azoxymethane-induced colon cancer in rats involved modulation of the colonic antioxidant system partly by flavonoids. Pharm. Biol., 2017, 55, 2102-2109.

32. - PAKSOY Z., GULESCI N., KANDEMIR M.K., CAGDAS D.G. : Effectiveness of levamisole and Tarantula cubensis extract in the treatment of teat papillomatosis of cows. Indian J. Anim. Res., 2015, 49, 704-708.

33. - PAN Y., RHEA P., TAN L., CARTWRIGHT C., LEE H.J., RAVOORI M.K., ADDINGTON C., GAGEA M., KUNDRA V., KIM S.J., NEWMAN R.A., YANG P. : PBI-05204, a supercritical CO₂ extract of Nerium oleander, inhibits growth of human pancreatic cancer via targeting the PI3K/mTOR pathway. Invest. New Drugs, 2015, 33, 271-279.

34. - RAJU J. : Azoxymethane-induced rat aberrant crypt foci: Relevance in studying chemoprevention of colon cancer. World J. Gastroenterol., 2008, 14, 6632-6635.

35. - REFAAT B., EL-SHEMI A.G., KENSARA O.A., MOHAMED A.M., IDRIS S., AHMAD J., KHOJAH A. : Vitamin D3 enhances the tumouricidal effects of 5-fluorouracil through multipathway mechanisms in azoxymethane rat model of colon cancer. J. Exp. Clin. Cancer Res., 2015, 34, 71.

36. - RICHTER PHARMA. : https://www.richter-pharma.at/product-theranekron-d6_301.htm, Accessed at: 04.06.2018.

37. - SARDARI K., KAKHKI E.G., MOHRI M. : Evaluation of wound contraction and epithelialization after subcutaneous administration of Theranekron® in cows. Comp. Clin. Pathol., 2007, 16, 197-200.

38. - SHWTER A.N., ABDULLAH N.A., ALSHAWSH M.A., ALSALAHI A., HAJREZAEI M., ALMAQRAMI A.A., SALEM S.D., ABDULLA M.A. : Chemoprevention of colonic aberrant crypt foci by Gynura procumbens in rats. J. Ethnopharmacol., 2014, 51, 1194-1201.

39. - SONMEZER M.C., TULEK N. : Biomarkers in bacterial infections and sepsis. Klimik Dergisi, 2015, 28, 96-102.

40. - TAKAYAMA T., MIYANISHI K., HAYASHI T., KUKITSU T., TAKANASHI K., ISHIWATARI H., KOGAWA T., ABE T., NIITSU Y.: Aberrant crypt foci: detection, gene abnormalities, and clinical usefulness. Clin. Gastroenterol. Hepatol., 2005, 3(Suppl 1), 42-45.

41. - TERZIC J., GRIVENNIKOV S., KARIN E., KARIN M. : Inflammation and colon cancer. Gastroenterology, 2010, 138, 2101-2114.

42. - UCHIDA E., CHAMBERS J.K., NAKASHIMA K., SAITO T., OHNO K., TSUJIMOTO H., NAKAYAMA H., UCHIDA K. : Pathologic features of colorectal inflammatory polyps in miniature dachshunds. Vet. Pathol., 2016, 53, 833-839

43. - WALY M.I., AL-RAWAHI A.S., AL-RIYAMI M., AL-KINDI M.A., AL-ISSAEI H.K., FAROOQ S.A., AL-ALAWI A., RAHMAN M.S. : Amelioration of azoxymethane induced-carcinogenesis by reducing oxidative stress in rat colon by natural extracts. BMC Complement Altern. Med., 2014, 14, 60.

44. - WOLDEMESKEL M., HAWKINS I., WHITTINGTON L. : Ki-67 protein expression and tumor associated inflammatory cells (macrophages and mast cells) in canine colorectal carcinoma. BMC Vet. Res., 2017, 13, 111, doi: 10.1186/s12917-017-1030-7.

45. - YALCIN A.S., ATTAALLAH W., YILMAZ A.M., AKTAN A.O. : Free radicals, whey proteins and colorectal cancer. Marmara Med. J., 2014, 27, 1-6.

46. - YARDIMCI C., YARDIMCI B. : Indolent ulcer in a cat. Ankara Univ. Vet. Fak. Derg., 2008, 55, 65-67.

47. - YAZAR E., TRAS B. : Free oxygen radicals, antioxidant enzymes and antibiotics. J. Turk Vet. Med. Assoc., 2002, 14, 42-44.

48. - YAZIHAN N., BAS A.L., ERMIS E., DEMIRCI S., UNEY K. : Increased glucose uptake and insulin binding activity of Nerium oleander in hepatocytes and adipocytes. Kafkas Univ. Vet. Fak. Derg., 2013, 19, 25-30.

49. - YOON H.Y., MANN F.A. : Bilateral pubic and ischial osteotomy for surgical management of caudal colonic and rectal masses in six dogs and a cat. J. Am. Vet. Med. Assoc., 2008, 232, 1016-1020.