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Biotechnic & Histochemistry
ISSN: 1052-0295 (Print) 1473-7760 (Online) Journal homepage: https://www.tandfonline.com/loi/ibih20
Effects of paclitaxel and resveratrol on blood
characteristics in rabbits
Sercan Kaya & Hasan Hüseyin Dönmez
To cite this article: Sercan Kaya & Hasan Hüseyin Dönmez (2020) Effects of paclitaxel and resveratrol on blood characteristics in rabbits, Biotechnic & Histochemistry, 95:3, 198-202, DOI: 10.1080/10520295.2019.1663557
To link to this article: https://doi.org/10.1080/10520295.2019.1663557
Published online: 01 Oct 2019.
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Effects of paclitaxel and resveratrol on blood characteristics in rabbits
Sercan Kaya aand Hasan Hüseyin Dönmez b
aBatman University, Health Services Vocational School, Medical Laboratory Program, Batman, Turkey;bSelçuk University, Veterinary Faculty, Department of Histology and Embryology, Konya, Turkey
ABSTRACT
We investigated the adverse effects of paclitaxel (PAC) on blood characteristics including the percentage of alpha naphthyl acetate esterase (ANAE)-positive peripheral blood lymphocytes, white cell count, number of neutrophil nuclear lobe and, erythrocyte diameter, and the protective effects of resveratrol (RES) on these characteristics. Male New Zealand rabbits were assigned to four groups. The control group (C) was given 40 ml normal saline, the PAC group was given 5 mg/ kg PAC in 40 ml normal saline, the RES group was given 4 mg/kg RES in 40 ml normal saline, and the PAC + RES group was given 5 mg/kg PAC + 4 mg/kg RES in 40 ml normal saline. All injections were performed intravenously once/week for 8 weeks. PAC caused an increased total percentage of lymphocytes and monocytes, which was associated with neutropenia. The PAC group showed a right shift in the lobulation curve of neutrophil nuclei together with a decreased percentage of ANAE-positive lymphocytes. RES reduced the percentage of ANAE-positive lymphocytes, slightly increased the percentage of neutrophil leukocytes and reduced the total lymphocyte count. Administration of RES combined with PAC prevented, while PAC induced, neutropenia and lymphocytosis, and increased the percentage of ANAE-positive lymphocytes.
KEYWORDS
blood characteristics; paclitaxel; rabbit; resveratrol
Paclitaxel (PAC) is an antiproliferative taxane agent used to treat cancer. It stabilizes microtubules at the anaphase stage of mitosis by altering alpha- and beta-tubulin polymerization. PAC inhibits both intimal hyperplasia and stenosis by inhibiting the growth, proliferation and migration of smooth muscle cells (Hoeven et al. 2005). Sgadari et al. (2000) reported that while PAC is an inhibitor of Bcl-2 anti-apoptotic activity and a microtubule stabilizer that is effective for treatment of some neoplasms; it also is effective in patients with Kaposi’s sarcoma, which has been shown to be associated with advanced human immunodeficiency virus (HIV) disease. PAC can be used either alone or in combination with other drugs to treat non-Hodgkin’s lymphoma (NHL) and multiple myeloma (MM) (Miller et al. 1998; Goss et al. 1999). Jazirehi and Bonavida (2004) reported that apoptosis increased in tumor cells when chemotherapeutics were combined. These investigators also reported that when administered in combination with PAC, resveratrol (RES) overcame the resistance of NHL and MM cells, and rendered them susceptible to PAC administration. Alpha-naphthyl acetate esterase (ANAE), a lysosomal enzyme, is used to differentiate T lymphocytes, B lymphocytes and monocytes from each other in tissue
and peripheral blood smears. ANAE is acquired during the advanced stages of T lymphocyte maturation. Like other esterases, ANAE participates in the cytotoxic functions of active T lymphocytes and the degradation of phagocytic material within macrophages (Sur et al.2005).
RES is a polyphenolic stilbene compound found particularly in grapes and wines (Li 2006). RES exerts its anticancer effects by inhibiting or activating various cellular targets that inhibit cell growth, induce apoptosis, block angiogenesis and prevent tumor invasion and metastasis (Espinoza et al. 2018). In addition to the potentiating interactions of RES with drugs used to treat metabolic disorders, such as metformin (Bruckbauer and Zemel 2013), RES has been reported to enhance the anti-tumor effects of various anti-neoplastic drugs used for hematological malignancies and to exert biological effects on circulating immune cells (Espinoza et al. 2017; Tripathi et al.2018; Warburton et al.2018).
Considering the clinical success achieved with new anticancer immunotherapies, especially using immune check point inhibitors, combining RES with immunotherapy is an attractive therapeutic approach that should be explored in preclinical studies (Espinoza et al.2018).
CONTACTSercan Kaya sercan.kaya@batman.edu.tr Batman University, Main Campus, Health Services Vocational School, Room 212, Kültür Mahallesi, Batman, Turkey
https://doi.org/10.1080/10520295.2019.1663557
We investigated the effects of RES alone, PAC alone, and the combination of RES and PAC on the percentages of peripheral blood T lymphocytes, number of leukocytes, nuclear lobulation of neutrophil leukocytes and changes in red blood cell volume.
Material and methods Animals
Our study was conducted at the research laboratory of Selçuk University Veterinary Faculty, Experimental Animal Unit with the approval of the Ethics Committee of Selcuk University Veterinary Faculty (Decision no. 2013/059). Thirty-two 24−36-week-old 2.5−3 kg male New Zealand rabbits were obtained from the Laboratory Animal Unit of Selçuk University Veterinary Faculty. Animals were assigned to four groups of eight. Control (C), PAC, RES and PAC + RES groups were injected intravenously with 40 ml normal saline, 5 mg/kg PAC in 40 ml normal saline, 4 mg/kg RES in 40 ml normal saline and 5 mg/kg PAC + 4 mg/kg RES in 40 ml normal saline, respectively. All injects were into the marginal ear vein (vena auricularis lateralis) once weekly for 8 weeks.
Blood
At the end of 8 weeks, blood samples were taken using a branule from the marginal ear vein and four peripheral smears were prepared for each animal. After air drying, two smears were used to identify peripheral blood ANAE-positive lymphocytes using the ANAE demonstration method described below (Özcan 2005). The remaining two peripheral smears were stained with May Grünwald-Giemsa (Konuk
1981) for calculating differential leukocyte counts, counting neutrophil nuclear lobes and measuring erythrocyte diameter. Counts and diameter measurements were performed using a Leica DFC 320 camera attached to a Leica DM 2500 light microscope (Heerbrugg, Switzerland).
For ANAE demonstration, peripheral smears were air dried and fixed in a glutaraldehyde-acetone solution (50% glutaraldehyde:50% acetone, pH 4.8, at -10 ºC) for 3 min. A working solution was prepared by adding ANAE substrate to hexosotized pararosaniline solution prepared in phosphate buffer adjusted to pH 5.8. The smears were incubated in this solution for 2 h (Özcan2005). After nuclear staining using 1% methyl green (Dönmez and Sur 2008), the smears were mounted with Entellan and examined using a light
microscope (DM 2500; Leica). Lymphocytes exhibiting brown cytoplasmic staining in the form of≥ 1 dot were considered positive, while other lymphocytes were considered negative (Figure 1). Means were calculated using 200 lymphocytes in each smear.
Statistical analysis
For discrete and continuous variables, descriptive statistics (mean ± SD and percentile) were calculated. The homogeneity of the variances, which is a prerequisite of parametric tests, was evaluated using Levene’s test. The assumption of normality was tested using the Shapiro-Wilk test. To compare differences between three and more groups, one-way analysis of variance was used when the parametric test prerequisites were fulfilled, and the Kruskal Wallis test was used when these prerequisites were not fulfilled. The Duncan method, which is a multiple comparison test, was used to evaluate the significant results for three and more groups. The data were evaluated using SPPS 25 (IBM Corp., 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY). Values for p≤ 0.05 were considered significant.
Results
The distribution of ANAE stained lymphocytes in the smears is shown inFigure 2. The percentage of ANAE stained lymphocytes was 65.87% for the control group, 51.62% for the PAC group, 61.12% for the RES group and 58.62% for the PAC + RES group.
In the PAC group, the number of neutrophils with three or four nuclear lobes increased compared to all other groups, while the number of neutrophils with two nuclear lobes was higher in all other groups (Figure 3).
Figure 1.ANAE demonstration of rabbits in the RES group. Arrow, ANAE-positive lymphocytes; arrowheads, ANAE-negative lymphocytes.
Our findings for the differential leukocyte count are presented inFigure 4. We found that in the PAC group the percentage of lymphocytes increased significantly (p < 0.05) and the neutrophil percentage decreased significantly (p < 0.05) compared to the other groups (Figure 4).
The mean diameter of erythrocytes was 6.56μm for the control group, 6.58μm for the PAC group, 6.53 μm for the RES group, and 6.73 μm for the PAC + RES group; the groups did not differ significantly from each other for this characteristic.
Discussion
The literature contains reports of peripheral ANAE-positive lymphocyte percentages of 59.3% in ostriches, 51.8% in turkeys (Ergun et al. 2004) and 54.4% in chickens (Dönmez et al.2002). Some reports indicate that the number of lymphocytes circulating in the peripheral blood may vary with age, sex and breed (Dönmez and Sur
2008). ANAE-positive lymphocyte percentages of 68% and 72.8% have been reported for adult female Wistar rats and young female Sprague-Dawley rats, respectively (Şimşek et al. 2007). Özcan (2005) reported a peripheral blood ANAE-positive lymphocyte percentage of 68.2% for Ankara rabbits, whereas we found the mean ANAE-positive lymphocyte percentage to be 65.9%. in New Zealand rabbits. We found that the percentage of ANAE-positive lymphocytes was 51.6% for the PAC group, 61.1% for the RES group and 58.6% for the PAC + RES group. These data show that while PAC reduced the T lymphocyte percentage, RES administered in combination with PAC almost completely counteracted this effect. Vicari et al. (2009) reported that PAC administration to mice decreased significantly the number of CD4-positive lymphocytes in the peripheral blood. Hakim et al. (1997) also reported significant reductions in the number of CD4-positive lymphocytes shortly after PAC administration to patients with breast cancer.
0 10 20 30 40 50 60 70
C. PAC. RES. PAC & RES
65.9±0.97a
51.6±1.10c
61.1±1.02b
58.6±0.94b
Figure 2.Distribution of ANAE-positive lymphocytes (%). Groups with different superscripts differ significantly (p < 0.05).
0 5 10 15 20 25 30 35 40 45
C. PAC. RES. PAC & RES
15.25±0.39a 10.25±0.51c 14.75±0.61a 13.12±0.51b 44.62±0.85a 39.37±0.89b 44.75±0.76a 44.87±0.68a 25.62±0.91b 30.62±0.74a 25.12±0.53b 28.62±0.60a 13.37±0.46b 18.62±0.94a 14.25±0.82b 12.5±0.39c 1.12±0.19 1.12±0.13 1.12±0.13 0.87±0.12 1 Lobe 2 Lobes 3 Lobes 4 Lobes 5 Lobes
We found that the total lymphocyte percentage in the differential leukocyte count was 59.15% for the control group, 67.06% for the PAC group, 57.53% for the RES group and 59.75% for the PAC + RES group. Melichar et al. (2001) investigated the effects of combined doxorubicin + PAC chemotherapy on the peripheral blood leukocyte phenotype in breast cancer patients. These investigators attributed the increased lymphocyte counts after chemotherapy or hormonal therapy to an indirect mechanism associated with reduction of the tumor mass and recovery after the tumor-induced immunosuppressive effect.
Bulitta et al. (2009) repored that the use of PAC regimens for cancer patients may cause neutropenia. Similarly, Demirci et al. (2014) reported that when patients with breast cancer were treated with PAC and docetaxel, significant febrile neutropenia developed. We found that the neutrophil percentage was 31.60% for the control group, 22.31% for the PAC group, 37.21% for the RES group and 31.31% for the PAC + RES group. Consistent with earlier reports (Shitara et al. 2010; Demirci et al. 2014), we observed that PAC caused a statistically significant decrease in the neutrophil count. Generally, mature neutrophils possess a nucleus with three or four lobes. Changes in nuclear lobulation may indicate changes in the general state of health of the organism (Carvalho et al. 2015). Nakoyama and Yamaguchi (2005) suggested that the formation of multilobed nuclei in granulocytes may result from a delay during the S phase of the cell cycle. It also has been suggested that microtubules constituting the cell skeleton during differentiation may play a role in nuclear segmentation (Campbell et al.1995). Hoeven et al. (2005) reported that PAC affects tubulin polymerization by stabilizing microtubules in neutrophils. We found that the
PAC group exhibited a rightward shift in the nuclear lobulation curve of neutrophils compared to the other groups. We found that the neutrophils were aged cells and that new neutrophil production had been suppressed based on the existing neutropenia. Lobulation of neutrophil nuclei in the PAC + RES group differed from the control group except for neutrophils with two lobes and exhibited a leftward shift in the nuclear lobulation curve compared to the PAC group, which indicates that RES reduced the inhibitory effect of PAC on microtubule polymerization.
A significant increase was detected in the number of neutrophils of the RES group. In the PAC + RES group, RES exhibited a preventive effect and PAC exhibited an inductive effect for neutropenia. Although the percentage of ANAE-positive lymphocytes decreased significantly in the PAC group compared to the other groups, the percentage of ANAE-positive lymphocytes in the PAC + RES group was close to that of the control group. If supported by future studies concerning dose and duration, and other antioxidants that may be used in combination with RES, it appears that RES administration could be useful for strengthening immunity and restoring blood characteristics to normal levels in patients treated with PAC.
Conflict of interest
The authors report no potential conflict of interest related to this research.
Funding
This research is part of Sercan Kaya’s Master’s thesis and was supported by the Selçuk University, Scientific Research Projects Coordination Unit under Grant number 14202004. 0 10 20 30 40 50 60 70
C. PAC. RES. PAC & RES 59.15±1.52b 67.06±1.29a 57.53±1.13b 59.75±1.03b 31.6±1.99a 22.31±1.14b 37.21±4.04a 31.31±1.05a 8±0.48b 9.5±0.52a 8.4±0.53b 7.5±0.53c 1±0.17ab 1±0.19ab 0.75±0.11b 1.31±0.13a 0.28±0.08 0.12±0.06 0.12±0.06 0.09±0.04 Lymphocytes Neutrophils Monocytes Eosinophils Basophils
Figure 4.Distribution of ANAE-positive lymphocytes (%). Groups with different superscripts differ significantly (p < 0.05).
ORCID
Sercan Kaya http://orcid.org/0000-0001-9014-2448 Hasan Hüseyin Dönmez http://orcid.org/0000-0003-4664-8489
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