Hepatic protective effect of grape seed proanthocyanidin extract against Gleevec-induced apoptosis , liver Injury and Ki 67 alterations in rats

1Pharmaceutical Science Department, Faculty of Pharmacy, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia, 2Pharmaceutical Practice Department, Faculty of Pharmacy, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia, 3Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 4Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt, 5Departement of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, Tanta, Egypt


INTRODUCTION
Chemotherapy has been used for cancer treatments where it destroys cancer cells or may stop cancer cells from spreading, slows the growth of cancer cells and may cause toxic effects in normal cells surrounded by cancer that can severely affect the life of patients, which may cause death (Tousson et al., 2014a,b,c;2016).Gleevec (imatinib) is an antineoplastic chemotherapeutic agent used in the treatment of chronic myelogenous leukemia, gastrointestinal stromal tumors, several other malignancies and hypereosinophilic syndrome (Cools et al., 2003;Nakajima, Toga, 2003;Droogendijk et al., 2006).The most common side-effects with Gleevec are weight gain, neutropenia, thrombocytopenia, anemia, headache, nausea, vomiting, diarrhea, dyspepsia, abdominal pain, oedema, skin rashes, muscle spasm and cramps, muscle and joint pain, and fatigue (Gini, Hessenm Minnaard, 2005).
Recently, several polyphenolic antioxidants derived from grape seeds have been implicated in cell protection (Bagchi et al., 2014;Zhu et al., 2014).Grape seed proanthocyanidin extract (GSPE) is a rich source of proanthocyanidins that are natural antioxidants, which are composed of various polyphenolic compounds and generally believed to protect against reactive oxygen species (ROS)-mediated myocardial ischemia/ reperfusion injury and apoptosis (Fan, Lou, 2004;Cuevas et al.,2011;Zhu et al., 2014).Proanthocyanidins, also called condensed tannins, are oligomers and polymers of monomeric flavonoids (flavan-3-ols), containing various amounts of catechin and epicatechin, widely distributed in the plant kingdom, and which appears in fruits, vegetables and seeds especially those extracted from grape seeds (Mouradov, Spangenberg, 2014;Malisch et al., 2015).Proanthocyanidins have become of great interest due to their biological properties, such as antioxidant, anti-inflammatory and anticancerigen properties, with further investigation of interest due to the potential use of proanthocyanidins in cancer prevention, in addition to their protective effects by reducing mitochondria damage and inhibiting cell apoptosis (Zhou, Raffoul, 2012).Therefore, the present study was conducted to examine the possible modifying effects of GSPE against liver injury and Ki67 alterations induced by Gleevec in male rats.
Experiments were performed on 40 male albino rats weighing 130-150 g and 9-10 weeks of age.The rats were kept in our Faculty animal house for one week before the experimental work, and were maintained on a standard rodent diet and water available ad libitum.

Animal treatments
After a week of acclimatization, the rats were equally divided into four groups: 1 st group: The control group included rats that did not receive treatment. 2 nd group: GSPE or a positive control group in which animals received orally GSPE by stomach tube a dose of 50 mg/kg body weight twice a week for four weeks according to Zhang et al. (2005).It was purchased from Sigma Company. 3 rd group: Gleevec group in which rats were injected intraperitoneally with Gleevec (1 mg/kg body weight/ twice a week) (Unistin 50 mL/50 mg vial Eimc united Pharmaceutical Badr City, Cairo, Egypt) for four weeks according to Prasad, Ramnarayan and Bairy (2010).4 th group: Co-treated group included rats that received GSPE (50 mg/kg B.W/ twice a week) orally for four weeks and injected simultaneously by Gleevec (1 mg/kg BW) twice a week for four weeks.

Determination of the P53 and Bcl2 protein levels
P53 protein was determined by Assay designs kit (catalog No.900-117) while Bcl-2 was determined by Assay designs kit package .According to the manufacturer's instructions, determining P53 and Bcl2 protein levels was performed by colorimetric procedures using human ELISA kit (Biospes, Chongqing, china) according to Findley et al. (1997) and Binabaj et al. (2015).

Histopathological examination
At the end of the experimental period, animals were seen to have fasted overnight and immediately after decapitation, rats were dissected, and liver from different groups were quickly removed and fixed in 10% neutral buffered formalin.After fixation, specimens were dehydrated, cleared in xylene and embedded in molten paraffin.Sections of 5 μm thickness were cut using a rotary microtome and stained with Ehrlich's haematoxylin and counterstained with eosin as a routine method after Bancroft and Stevens (1990).

Statistical analysis
Data were expressed as mean values ± SE and statistical analysis was performed using one way ANOVA to assess significant differences among treatment groups.The criterion for statistical significance was set at p<0.01 for the biochemical data.All statistical analyses were performed using SPSS statistical version 16 software package (SPSS ® Inc., USA).

Gleevec induced apoptosis
Table I shows the concentration of apoptotic protein P53 and anti-apoptotic protein Bcl2 in liver tissue of different groups.P53 levels were significantly increased in treated rats with Gleevec when compared with control group.In contrast, prophylactic treatment with Gleevec and GSPE prevented Gleevec induced elevations in P53 levels compared to the control group (Table I).On the other hand, the anti-apoptotic protein Bcl2 levels in liver tissue were significantly (P<0.01)decreased in Gleevec when compared with control group; while, prophylactic treatment with Gleevec and GSPE prevented Gleevec induced depletion in Bcl2 levels compared to the control group (Table I).

Effect of GSPE on liver histopathology
Liver sections of control and GSPE groups exhibited the normal architecture of the hepatocytes with prominent round, vesicular basophilic nuclei and eosinophilic cytoplasm, and a few spaced hepatic sinusoids arranged in-between the hepatic cords (Figure 1A and B).Liver sections of Gleevec group exhibited marked cellular infiltrations, moderate atrophied vacuolar degeneration hepatocytes, numerous apoptotic cells with deep eosinophilic cytoplasm and small deeply stained pyknotic or fragmented nuclei, as well as marked dilation or congestion in central and portal veins (Figure 1C).Liver sections in treated Gleevec with GSPE exhibited a mild degree of improvement in hepatocytes that exhibits moderate vacuolated hepatocytes with mild cellular infiltrations and moderate congestion in central and portal veins (Figure 1D).

Effect of GSPE on liver Ki67 immunohistochemical changes
T h e d e t e c t i o n a n d d i s t r i b u t i o n i n K i 6 7 immunoreactivity (Ki67-ir) in liver sections in the different groups are shown in Figures 2A-2D.Strong positive reaction for Ki67-ir (grade 4) in hepatocyte nuclei was observed in liver section in control and GSPE groups (Figures 2A and B).In contrast, mild to faint positive reactions were detected for Ki67-ir (grade 1) in the liver sections in Gleevec rats group (Figure 2C).On the other hand, moderate positive reactions for Ki67-ir (grade 2) were observed in liver sections of treated Gleevec with GSPE (Figure 2D).

DISCUSSION
Chemotherapy involves the use of chemical agents to stop the growth and elimination of cancer cells even at distant sites from the origin of primary tumor.However, chemotherapy does not distinguish between cancer and normal cells, and eliminates not only the fast-growing cancer cells but also other fast-growing cells in the body, including blood cells.Although a number of studies have demonstrated some side-effects of the chemotherapeutic drugs, the current work is aimed at studying the possible modifying effects of GSPE extract against liver injury, apoptosis and changes in proliferating markers Ki67 induced by Gleevec in the liver tissues in male rats.
When cells are exposed to external damage stimuli or injury, they activate the regulation of expression of these genes.P53 tumor suppressor protein is a transcription factor that regulates the DNA repair and apoptosis, while Bc1-2 gene is an anti-apoptotic protein, which has important roles in regulating cell survival (Tousson et al., 2014a;2016).An inverse correlation was found between the expression of Bcl-2 and p53 proteins in our results.In the current study, injection with Gleevec for four weeks induced elevations in P53 and depletion in Bcl2 levels in liver tissues compared to the control group.In contrast, treatment of rats with Gleevec and GSPE significantly decreased the P53 and increased the Bcl2 levels when compared with Gleevec group.The results from study agreed with Tousson et al. (2014a;2016) who reported that Amethopetrin induced apoptosis in lung and heart rats and L-carnitine treatment improved these alternations.
In the current study, a moderate atrophied vacuolar degeneration hepatocytes, marked cellular infiltrations, and marked dilation or congestion in central and portal veins were observed in liver sections after Gleevec injection.Our histopathological results showed that treatment of rat liver with GSPE showed a mild degree of improvement in hepatocytes in liver sections.Our results agree with Kart et al. (2010) and Basuony et al. (2015) who reported that chemotherapy drugs induces hepatotoxicity, oxidative injury and cellular abnormalities in liver.Similar findings were reported by El-Sayyad et al. (2009) and Abdel-Wahhab et al. (2014) who revealed that many hepatocytes showed marked degeneration in hepatic cords in addition to karyomegaly and pyknotic nuclei indicating apoptosis after treatment with chemotherapy.
The most widely used proliferation-associated marker is Ki-67, which is a nuclear antigen present only in proliferating cells (Lebe et al., 2007).In the current study, a significant decrease in Ki67 was observed in liver sections after Gleevec injection when compared with control.This current result is in harmony with Leitão et al. (2011) who reported that MTX treatment significantly decreased the numbers of Ki-67-positive cells.Although the numbers of Ki-67-positive cells had a significant decrease in the Gleevec group in the present study, this decrease was not observed in the co-treated Gleevec with GSPE groups.

FIGURE 1 -
FIGURE 1 -Photomicrographs of rat liver sections stained by HE; (A&B) Rat testes in control (G1) and GSPE (G2) groups showed normal architecture of the hepatocytes (hp) with central vien (CV); (C) Liver sections of Gleevec group (G3) exhibited marked cellular infiltrations (White arrows), moderate atrophied vacuolar degeneration hepatocytes (arrow heads), and many of apoptotic cells; (D) Liver sections treated Gleevec with GSPE (G4) exhibited a mild degree of improvement in hepatocytes that exhibits moderate vacuolated hepatocytes (arrow heads) with mild cellular infiltrations (White arrows) and moderate congestion in central and portal veins.

FIGURE 2 -
FIGURE 2 -Photomicrographs of liver sections stained with KI67-ir in the different groups; (A&B) Liver section in control and GSPE groups showed strong positive reaction (arrows) for Ki67-ir (grade 4) in hepatocyte nuclei; (C) Liver sections in Gleevec rats group showed mild to faint positive reactions for Ki67-ir; (D) Liver sections in treated Gleevec with GSPE showed moderate positive reactions (arrows) for Ki67-ir.

TABLE I -
The apoptotic protein (P53) and the antiapoptotic protein (Bcl 2 ) levels in rat liver tissues in different group