Effects of Tarantula cubensis extract and Capecitabine in Colorectal Cancer rats / Canbar et al. __________________________________
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INTRODUCTION
The incidence of colorectal cancer (CRC) is the third–highest of
all cancers [1]. CRC is also found in Veterinary Medicine [2]. Unlike
other cancer types, mutations in tumor suppressor genes (TSG)
have been reported in CRC [3]. Moreover, CRC arises due to defects
in oncogenes, TSG, and genes related to deoxyribose nucleic acid
(DNA) repair mechanisms [4]. Capecitabine is a new oral adjuvant
and palliative Fluoropyrimidine prodrug approved by The United
States Food and Drug Agency (FDA) in 1998 that inhibits Thymidylate
synthase [5] and is used to treat various cancer types. It has been
reported that Capecitabine is converted to 5–Fluorouracil (5–FU) when
prepared in oral formulation [6] with a recommended dose of 2,500
mg·m
-2
administered for 14 out of every 21 days [7]. Capecitabine is
converted to the active form after a three–step enzymatic activation
process. The enzyme Cytidine deaminase, which plays a role in the
catabolism of the drug in rats (Rattus norvegicus), is at a lower level
than in monkeys (Cnomolgus monkeys) and humans [8]. In addition,
the enzyme activity and plasma concentrations of its metabolites
have been reported to decrease with repeated applications of
Capecitabine [9]. Fluorodeoxyuridine monophosphate, which is
formed by the enzymatic conversion of 5–FU by Thymidine kinase,
is reported to inhibit Thymidylate synthetase, which is the rate–
limiting step in Thymidine synthesis. In addition, without Thymidine,
desoxi rribonuclec acid (DNA) synthesis is impaired, and cellular death
occurs. 5–FU is also converted to Fluorouridine Triphosphate (FUTP),
the antimetabolite of 5–FU, by Thymidine Phosphorylase (dThdPase),
and FUTP binds to RNA and instructs the cell to undergo apoptosis
[10]. Interestingly the dThdPase enzyme is found at higher levels in
cancerous tissues than in normal tissues, so Capecitabine has a more
effective and safer prole than 5–FU [11].
Tarantula cubensis alcoholic extract (TCAE) is a homeopathic
product used in Veterinary Medicine, where it is generally used to
treat conditions such as gangrene, septicemia, and toxemia [12].
Moreover, it is reported to increase apoptosis in cancer cells in vitro
via the caspase–3 (Cas–3) pathway [13] and to cause clinically positive
effects in caninen (Canis lupus familiaris) mammary tumors [14] via
apoptotic pathways [15]. It has been successfully used to treat canine
oral papilloma [16] and reported to reduce Bcl–2 and Ki–67 gene
expression in canine mammary adenocarcinoma [17], as well as reduce
aberrant crypt foci (ACF) and polyp formation in colon cancer [18].
In the last stage of apoptosis, caspases, which degrade vital
intracellular proteins, are activated. Bcl–2 is antiapoptotic, and
Bax is proapoptotic. It has been reported that caspase–9 activates
effector caspases [3, 6, 7] that degrade vital cellular proteins and
provide cellular destruction [19]. ACF is observed by staining the
colon tissue of CRC patients with Methylene blue. It has also been
found to be important for early CRC diagnosis [20]. ACF has been
identied as an important parameter in experimental CRC studies
where it is indicative of colon carcinogenesis [21].
TCAE [13] and Capecitabine [6] are individually effective in some
types of cancer. It has been hypothesized that the combined effects
of TCAE and Capecitabine on mitochondrial dysfunction in apoptosis
and ACF in rats with CRC would increase survival compared with
monotherapy. In this study, we determined the effects of combined
and single Capecitabine and TCAE treatment on ACF score and
expression of Bcl–2, Bax, and Cas–3 in rats with CRC.
MATERIALS AND METHODS
Animal material
This study used 96 male Wistar Albino rats (12–16 weeks old,
220–250 g obtained from Selcuk University Experimental Medicine
Application and Research Center, Konya, Turkey. Study protocol
was approved by ethic committee (Ethic No:2019–32). The rats
were randomly divided into eight groups with 12 animals in each
group: Healthy control (C), CRC control (CC), healthy with TCAE (C +
TCAE), CRC with TCAE (CRC + TCAE), healthy with Capecitabine (C +
Capecitabine), CRC with Capecitabine (CRC + Capecitabine), healthy
with TCAE and Capecitabine (C + TCAE+ Capecitabine), and CRC with
TCAE and Capecitabine (CRC + TCAE + Capecitabine). Azoxymethane
(AOM; 15 mg·kg
-1
, intraperitoneal injection (IP), administered twice in
a 1–week interval; Sigma–Aldrich, Germany) was administered to all
CRC groups [18, 22]. The rats in the TCAE groups were administered
TCAE (Theranekron D6 inj; Richter pharma AG, Austria) via IP at a dose
of 0,2 mL/rat for 4 weeks, with 3 days intervals, starting from the 15th
week [18, 23]. Capecitabine (Kapeda tablet, Kocak Farma, Istanbul,
Turkey) groups were orally (PO) administered Capecitabine daily for
30 days at 40 mg·kg
-1
(SID) starting in the 15th week. In the combined
treatment groups, both drugs were administered simultaneously with
the dose and administration method indicated for the single treatment
groups. Rats were sacriced by cervical dislocation one hour after
the last injection using Ketamine (95 mg·kg
-1
, subcutaneous –SC–)
and Xylazine (5 mg·kg
-1
, SC) anesthesia. After the colon tissue was
opened longitudinally and washed with physiological saline, tissue
samples were taken from the proximal, median, and distal regions
and immediately frozen in liquid nitrogen before being stored at –80°C
(Haier, DW–86L628, China) until needed for real–time polymerase chain
reaction (RT–PCR) analysis. The remaining tissue was xed with 10%
formaldehyde for pathological examination.
Molecular analysis
Tissues from six animals from each group were chosen randomly for
RT–PCR analysis. Equal amounts of samples taken from the proximal,
distal, and median portions of the colon were used to represent
the entire colon. Tissues were isolated with an Ribonucleic acids
(RNA) Isolation Kit (Biobasic; Markham, ON, Canada). The A260:A280
ratio was determined with a Total RNA 2000/2000 cycl (c) Nanodrop
Spectrophotometer (Thermo Fisher Scientic; Waltham, MA, USA).
All RNA samples were treated with DNase I (Thermo Fisher Scientic;
Waltham, MA, USA) to remove DNA contamination. Complementary
DNA (cDNA) was synthesized using the iScriptcDNA synthesis kit
(Bio–Rad, Hercules, CA, USA) according to the manufacturer’s
recommended protocol and stored at –20°C until required. The mRNA
information and sequences of the primers used to amplify the target
genes (Bax), (Bcl–2), and (Cas–3) housekeeping gene (GAPDH) are
presented in TABLES I and II, respectively [30, 45, 46]. In addition,
the mRNA and cDNA sequences of the genes were checked with
The National Center for Biotechnology Information (NCBI) GenBank
database (http://www.ncbi.nlm.nih.gov), and the sequences of their
primers were checked with NCBI’s Primer Blast (http://www.ncbi.nlm.
nih.gov/tools/) and the Oligo7 primer design program. The designed
PCR primers were synthesized by Oligomer (Ankara, Turkey).