S phase cell cycle transition in N-nitrosodiethylamine-induced experimental rat hepatocellular carcinoma

Toxicology Letters 223 (2013) 60–72

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Thymoquinone inhibits cell proliferation through regulation of G1/S phase cell cycle transition in N-nitrosodiethylamine-induced experimental rat hepatocellular carcinoma Subramanian Raghunandhakumar a , Arumugam Paramasivam b , Selvam Senthilraja c , Chandrasekar Naveenkumar a , Selvamani Asokkumar a , John Binuclara a , Sundaram Jagan a , Pandi Anandakumar a , Thiruvengadam Devaki a,∗ a

Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, India Department of Genetics, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, India c Center for Biotechnology, Anna University, Chennai 600 025, India b

h i g h l i g h t s • • • •

TQ inhibits cell proliferation. TQ down regulates the expression of proteins controlling the G1/S phase of cell cycle. TQ treatment greatly reduced liver injury markers and tumor markers. Treatment with TQ was greatly reduced the hyper AgNORs expression in NDEA induced hepatocellular carcinoma.

a r t i c l e

i n f o

Article history: Received 29 May 2013 Received in revised form 16 August 2013 Accepted 22 August 2013 Available online 3 September 2013 Keywords: Thymoquinone NDEA Proliferation G1/S cell cycle transition Hepatocellular carcinoma

a b s t r a c t Dysregulated cell proliferation and tumorigenesis is frequently encountered in several cancers including hepatocellular carcinogenesis (HCC). Thus, agents that inhibit cell proliferation and restrain hepatic tumorigenesis through cell cycle regulation have a beneficial effect in the treatment of hepatocellular carcinogenesis. The present study was aimed to investigate the efficacy of thymoquinone (TQ), an active compound derived from the medicinal plant Nigella sativa, on N-nitrosodiethylamine (NDEA) [0.01% in drinking water for 16 weeks]-induced hepatocarcinogenesis in experimental rats. After experimental period, the hepatic nodules, liver injury markers and tumor markers levels were substantially increased in NDEA induced liver tumors in rats. However, TQ (20 mg/kg body weight) treatment greatly reduced liver injury markers and decreased tumor markers and prevented hepatic nodule formation and reduced tumor multiplicity in NDEA induced hepatic cancer bearing rats and this was evident from argyrophilic nucleolar organizer region (AgNORs) staining. Moreover, the uncontrolled cell proliferation was assessed by specific cell proliferative markers [proliferating cell nuclear antigen (PCNA) and Ki67] by immunofluorescence, immunoblot and analysis of mRNA expression. Simultaneously, we assessed the activity of TQ on G1/S phase cell cycle regulation with specific cell cycle proteins (p21WAF1/CIP1 , CDK4, Cyclin D1 and Cyclin E) by immunoprecipitation in experimental rats. Treatment with TQ significantly reduced the detrimental alterations by abrogating cell proliferation, which strongly induced G1/S arrest in cell cycle transition. In conclusion, our results suggest that TQ has a potent anti proliferative activity by regulating the G1/S phase cell cycle transition and exhibit a beneficial role in the treatment of HCC. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

∗ Corresponding author at: Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India. Tel.: +91 44 22202736. E-mail address: [email protected] (T. Devaki). 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.08.018

Hepatocellular carcinoma (HCC) is the most common primary malignancies of the liver and the third leading cause of cancer death worldwide (Tsai and Chung, 2010). More than 80% of the new cases are detected mostly in developing countries of Asia and Africa. However, the incidence rate is 2–3 times higher in developing countries, and globally it has become a fastest growing cause of

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Fig. 1. Chemical structure of thymoquinone (a) and N-nitrosodiethylamine (b).

cancer death (Jemal et al., 2010; Sohal and Sun, 2011; Thomas et al., 2011). The vast majority of HCC cases are attributed to cirrhotic liver associated with viral hepatitis (B and C), alcohol, obesity, iron overload, and mainly due to dietary carcinogens, including aflatoxins and nitrosamines (Perz et al., 2006). N-nitrosodiethylamine (NDEA) (Fig. 1b) is widespread in the environment, and it is present in foods, beverages, tobacco smoke, agricultural chemicals, cosmetics, and industrial pollution. These are the major risk factors of liver diseases, and their endogenous formations causes a wide range of tumors, and are hazardous to human health NDEA is reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity in experimental animal models (Perz et al., 2006; Brown, 1999; Reh and Fajen, 1996; Smith et al., 1997, 2001). It is known to cause perturbations in the nuclear enzymes involved in DNA repair/replication (Ramakrishnan et al., 2006). The reactive oxygen species (ROS) formation occurs during the metabolic biotransformation of NDEA and leads to carcinogenesis by upregulation of biochemical, intracellular signaling pathways, and gene expression. One of the most promising applications in the emerging field of cancer treatment is to discover the adjuvant or palliative therapy for solid tumors with negligible cytotoxicity to the normal cells. Hence, the identification of novel compound preferentially from plant origin have been shown to be promising chemotherapeutic agent. Thus bioactive compound provide a novel opportunity to improve the existing standard of care for HCC and other cancers (Newman, 2008). Thymoquinone (TQ) (Fig. 1a) is a major bioactive ingredient isolated from Nigella sativa and has been reported for its antiinflammatory, antioxidant, and anti-neoplastic effects both in vitro and in vivo (Gurung et al., 2010; Gali-Muhtasib et al., 2006; Li et al., 2010). Moreover, TQ could act as a free radical, superoxide radical scavenger and conserving the activity of various antioxidant enzymes. In recent studies, anti-proliferative and therapeutic effect of TQ has been reported against a wide variety of cancer, including breast cancer, ovarian cancer (Shoieb et al., 2003), colorectal cancer (Gali-Muhtasib et al., 2004), pancreatic cancer (Worthen et al., 1998), osteosarcoma (Roepke et al., 2007), leukemia (ElMahdy et al., 2005), fibrosarcoma and lung cancer (Kaseb et al., 2007), and squamous cell carcinoma (Das et al., 2012). Recently, Li et al. have reported that TQ inhibits cell proliferation and induces apoptosis in multiple myeloma cells (Li et al., 2010). We have previously reported that anticancer activity of TQ in mouse neuroblastoma (Neuro-2a) cells through caspase-3 by down regulation of XIAP (Paramasivam et al., 2012). In animal models, TQ inhibited the forestomach tumors and enhanced the anti-tumor activity in pancreatic cancer (Salem, 2005). The exact mechanisms of TQ in inhibiting cell proliferation and how it restrained the tumor growth are yet to be studied. Cell cycle is regulated by protein complexes composed of cyclins and cyclin-dependent kinases (CDKs). Aberrant activation of cyclins and CDKs has been observed in numerous primary tumors which correlate with failure of cell cycle control which resulting in uncontrolled proliferation (King and Cidlowski, 1998). Several studies

Fig. 2. Schematic representation of the experimental protocol involving NDEA exposed experimental rat hepatocellular carcinogenesis.

documented that dysregulation of cell cycle causes uncontrolled proliferation and contribute to the tumor growth and neoplastic transformation in various malignancies, including breast, osteosarcoma, colon cancer and hepatocellular carcinoma (Woo et al., 2012). Therefore, inhibition of G1/S phase cell cycle transition by suppressing the Cyclin D1/CDK4 protein may block the development of carcinogenesis. This present study was attempted to explore the antiproliferative efficacy of TQ through cell cycle arrest at G1/S phase against NDEA induced HCC. 2. Materials and methods 2.1. Experimental animals and diet Pathogen-free adult male Wistar strain albino rats (Rattus norvegicus) weighing about 150–180 g were obtained from The King Institute, Chennai, India. The animals were acclimatized to standard laboratory conditions including a controlled environment at 24 ± 1 ◦ C and 50 ± 10% relative humidity with the alternating 12:12-h dark–light cycle for 1 week before the beginning of the study. The animals were fed a commercial pelletted diet (M/s Hindustan foods Ltd., Bangalore, India) and provided drinking water ad libitum. All the experiments were designed and conducted according to the ethical norms approved by Institutional animal ethics committee guidelines (IAEC No. 01/086/09) regulated by the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. 2.2. Chemicals and their sources NDEA and TQ were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies for PCNA, Ki67, p21WAF1/CIP1 , CDK4, Cyclin D1 and Cyclin E were purchased from Santa Cruz biotech, USA. All other chemicals used were purchased from Bio Basic (USA), Genei (Bangalore) and Sisco Research Laboratories (Mumbai) India. 2.3. Experimental design A schematic representation of the experimental protocol is given in Fig. 2. Following an acclimatization period of 1 week with standard basal diet, rats were randomly divided into five groups (10 animals per group) based on a power analysis. Group I is normal control; Group II is NDEA alone (Induced); Group III is TQ alone; Group IV is Preventive treatment (Pre treatment = TQ + NDEA); Group V is Curative/Post treatment (NDEA + TQ); Groups II and IV were given 0.01% NDEA in drinking water for 16 weeks and Group V for 11 weeks to induce HCC. TQ at a concentration of 20 mg/kg body weight was administered orally for weekly 3 alternative days to rats of Group IV, for two weeks before the experiment, and to rats of Group V for last 5 weeks of the experiment as per duration of treatment schedule. Food and water intake and behavioral changes were monitored every 2 weeks. At the end of the experimental period rats were fasted overnight and the body weight of each rat was monitored. Then the rats were anaesthetized and sacrificed by decapitation. The relative liver weight was calculated as the percentage ratio of liver weight to the body weight and stored at −20 ◦ C for further analysis.

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Table 1 List of primer pair sequences and amplicon sizes used in the study. Primer

Direction

Sequence

Amplicon size (bp)

NCBI accession number

PCNA

fwd rev

5 -CGTCGCAACTCCGCCACCAT-3 5 -GTTCACGCCGCCCGAACTGA-3

126

NM 022381.3

Ki67

fwd rev

5 -ATTCAGGCCCTGCGAAGCCG-3 5 -GCGTTGAAGGTAGGTGCCCCA-3

153

NM 139186.2

␤-Actin

fwd rev

5 -GCGTCCACCCGCGAGTACAA-3 5 -CGACGACGAGCGCAGCGATA-3

100

NM 031144.2

2.4. Evaluation of hepatic nodules

2.10. Confocal microscopic analysis of PCNA and Ki67

The rat liver was perfused with heparinized saline and subsequently excised, rinsed with ice-cold phosphate buffer saline (pH 7.4) to flush out any remaining blood and blotted dry on a filter paper. The dried liver was then photographed. Each liver was examined macroscopically on the surface as well as in 3-mm cross-section for gross visible hepatocyte nodules. The nodules were measured approximately to obtain an average diameter of the nodule and they were counted and categorized into three groups (i.e., ≥3, <3 to >1, and ≤1 mm) based on their respective diameters.

The immunofluorescence detection of proliferating cell nuclear antigen (PCNA) and Ki67, the cell proliferative markers of solid tumors were performed and observed using laser confocal microscopy in ∼3–5-␮m thick liver sections. The tissue sections were deparaffinized in xylene at 60 ◦ C for 15 min each and hydrated through graded series of alcohol. The hepatic sections were incubated in 10 mM sodium citrate buffer (pH 6.0) for 10 min at 80 ◦ C in microwave oven for antigen retrieval, then allowed to cool at room temperature. Following a 5-min wash with 1x Tris buffered saline/0.1%Tween-20 (TBST), blocked with TBST/5% BSA in room temperature for 1 h, it was incubated overnight with primary antibodies obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA (1:500 for PCNA mouse monoclonal and 1:100 for Ki67 rabbit polyclonal) at 4 ◦ C in a humidified chamber. After being washed with TBST, the sections were incubated with anti mouse and anti rabbit fluorescence isothio cyanate (FITC) conjugated secondary antibody (Bangalore Genei, India), diluted 1:50 with TBS and incubated in dark for 2 h at room temperature. The FITC-labeled secondary antibody obtained from Genei, Bangalore, India, was used. A chromogenic reaction was performed and the hepatic sections were counterstained with 100 ␮g/ml propidium iodide (PI) at room temperature for 30–45 min and then covered with mounting medium. Images were acquired under identical exposure conditions using laser scanning confocal microscopy (Leica TCS-SP2 XL, Germany).

2.5. Preparation of liver homogenate After cervical decapitation, a midventral incision was made on the experimental animals (using probe, scissors and finger) up to the entire length of the abdominal cavity till the diaphragm. Then a lateral incision was made of the rib cage, and at the hips, cutting deep enough to reach the body cavities. The blood was directly withdrawn by cardiac puncture and allowed to clot for 1 h before centrifugation at 4000 rpm for 15 min at 4 ◦ C to separate the serum. The liver was gently excised from underlying tissue with a blunt probe or careful cutting. The liver tissue was then washed with ice cold 0.1 M phosphate buffer saline (PBS, 1:9), pH 7.4, and homogenized with lysis buffer (1% NP40, 50 mM Tris pH 7.4, 150 mM NaCl). Homogenates were then centrifuged for 5 min at 14,000 rpm at 4 ◦ C and supernatants were used as whole protein extract. Isolated proteins were quantified using Lowry et al. both homogenates and serum were taken for the analysis as described in the later section. 2.6. Liver pathophysiological markers Serum was separated and levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase (ACP), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma glutamyltransferase (GGT), 5 -nucleotidase (5 -NT) and total bilirubin were estimated using an automated biochemical analyzer (Beckman Coulter, USA). The total protein in the serum was estimated by the method of Lowry et al. (1951). 2.7. Liver histology According to the procedure of Zhang et al. (2004), the whole liver (all four lobes) from each rats was fixed in Tellyesniczky’s solution (90% ethanol, 5% glacial acetic acid, 5% formalin overnight, followed by 70% ethanol). Fixed liver tissues were washed overnight, dehydrated through graded alcohols and embedded in paraffin wax. Serial sections of about 3–5 mm thickness were stained with hematoxylin and eosin (H and E) for histological examinations. 2.8. Argyrophilic nucleolar organizer regions (AgNORs) staining Argyrophilic nucleolar organizer regions (AgNORs) staining was performed according to the method of Ploton et al. (1986). Briefly, the sections were dewaxed in xylene and hydrated through decreasing concentration of ethanol to deionised water. The AgNOR solution was freshly prepared by dissolving gelatin at a concentration of 2 g/dl in 1 ml/dl aqueous formic acid. This solution was added to 50 g/dl aqueous silver nitrate solution (1:2, v/v). This final solution was then immediately poured on to the slides, which were left in the dark at room temperature for 45 min. The silver colloid was washed from the section with deionised water and the sections were dehydrated through a graded series of ethanol to xylene. For quantification, a mean of 10 different areas of sections was chosen to determine the homogenous AgNOR quantification throughout all groups at least 500 cells were counted, the quantification was performed in well-preserved cells and count was obtained by enumeration of both intra- and extra-nucleolar AgNOR dots; the AgNOR dots were easily identified as black points within the nuclei.

2.11. Immunoblot analysis The samples were boiled for 5 min before loading onto gels and the equal amount of protein (60 ␮g) were loaded and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was then transferred electrophoretically to polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were blocked with TBST/5% non-fat milk (blocking buffer) for 1 h at room temperature and blots were probed with following antibodies: mouse monoclonal PCNA, rabbit polyclonal Ki-67, rabbit monoclonal p21WAF1/CIP1 , rabbit polyclonal Cyclin D1, rabbit polyclonal Cyclin E, rabbit polyclonal CDK4 [Santacruz Biotech, USA]. Following incubation, immunoreactive bands were detected by incubating with respective HRP conjugates. Primary and secondary antibodies were diluted in TBST with 1% BSA (w/v). The bound antibodies were visualized using an enhanced chemiluminescence detection kit (Millipore, USA) in Chemi Doc image scanner from Bio Rad. The band intensity was quantified by Quantity One software (Bio Rad, USA). The membranes were stripped and reprobed for ␤-actin (1:5000) as an internal control. 2.12. Isolation of RNA and Reverse Transcriptase-PCR The total RNA was isolated by using Tri Reagent (Invitrogen). Total RNA (1 ␮g) from each sample was reverse transcribed using a commercial iScript cDNA synthesis kit according to the manufacturer’s protocol, Bio-Rad, USA. The details of the primers used, number of cycles, and size of the PCR amplified products are listed in Table 1. The PCR products were resolved by electrophoresis through a 2% agarose gel and stained with ethidium bromide. The band intensity was measured using a Gel Doc image scanner (Bio-Rad, USA), and quantified by Quantity One Software (Bio-Rad, USA). 2.13. Statistical analysis Statistical significance was evaluated with SPSS/10 software. Hypothesis testing methods included one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test. p values < 0.05 were considered to indicate statistical significance. All the results were expressed as mean ± SD for ten animals in each group.

3. Results 2.9. Liver tumor markers The quantitative estimation of liver tumor markers ␣-feto protein (AFP) and carcinoembryonic antigen (CEA) was monitored based on solid phase enzyme linked immunosorbent assay (ELISA) in serum using SIEMENS fully automated ADVIA Centaur, Bayer, USA.

3.1. General observations During the experimental period the rats tolerated the oral administration of NDEA and/or TQ feeding well and there were no

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Table 2 Effect of TQ on body weight and liver weight of control and NDEA-intoxicated groups of rats. Group (n = 10)

Treatment

Initial body weight (g)

I II III IV V

Control NDEA TQ TQ + NDEA NDEA + TQ

168 165 165 163 164

± ± ± ± ±

9.24 8.58 8.58 7.82 8.2

Final body weight (g) 270 155 265 225 205

± ± ± ± ±

15.93 6.35a 15.37 12.83b , c 12.3b

Liver weight (g) 7.1 10.2 7.21 9.1 9.87

± ± ± ± ±

0.25 0.83a 0.27 0.69b , c 0.77b

Relative liver (liver/100 g body weight) 3.48 5.89 3.68 4.01 4.78

± ± ± ± ±

0.11 0.29a 0.13 0.19 b , c 0.24b

Results are expressed as mean ± SD (n = 10). a Statistical significance at p < 0.05 compared with Group I. b Statistical significance at p < 0.05 compared with Group II. c Statistical significance at p < 0.05 compared with Group V.

clinical signs of toxicity related death in experimental groups, i.e., 16 weeks. There was a moderate difference in the growth rates of control and experimental rats. The data on mean body weight, liver weight, and relative liver weight of experimental rats after experimental period were summarized in Table 2. There was a slight decrease in the final body weight of NDEA-administered (Group II) rats as compared with the control (Group I) rats; conversely, treatment with TQ maintained the normal body weights in TQ pre- and post-treated (Group IV and Group V) rats as compared with control (Group I) rats, suggesting that TQ had no adverse effect on the growth response of the rats. The average liver weight of NDEA-administered (Group II) rats was significantly (p < 0.05) increased as compared with control (Group I) rats. A similar observation was observed for the liver weights between TQ pre- and post-treated (Group IV and Group V) rats. The relative liver weight in NDEA-administered (Group II) rats was found to be significantly (p < 0.05) higher than that of control (Group I) rats. The TQ pre- and post-treated (Group IV and Group V) rats relative liver weight was significantly (p < 0.05) lowered when compared with NDEA-induced (Group II) rats. However, there was no significant difference in liver weights and relative liver weights on TQ preand post-treated (Group IV and Group V) rats as compared with control (Group I) and TQ alone (Group III) rats. 3.2. TQ inhibits NDEA-induced nodule growth Fig. 3 and Table 3 summarize the gross observation of liver, tumor incidence, and their multiplicity of control and experimental rats. There were no visible hepatocyte nodules in the livers of normal control (Group I) rats (Fig. 3a), whereas the NDEA-administered (Group II) rats (Fig. 3b) showing abnormal appearance included a number of well-developed grayish-white foci or nodules of liver tumors in many lobes, which indicate the initiation of tumor nodules. In contrast, the TQ pre and post-treated (Group IV and Group V) rats (Fig. 3d and e) showed significantly reduced incidence of tumor nodules and multiplicity when compared with NDEAadministered (Group II) rats. Table 3 also shows tint in the size of visible nodules distribution, mean nodular volume, and nodular volume as per the percentage of the experimental group rats. These results suggest that suppression of nodule growth and hastening of nodule regression by TQ as observed in the current study could be viewed as a pivotal step for inhibition of tumor growth.

damage. However, the status of these marker enzymes in the TQ alone-administered (Group III) rats does not exhibit any significant alterations; which indicates the non-toxic nature of TQ. However, the TQ (pre and post)-treated rats showed significant (p < 0.05) decrease in the activities of these enzymes when compared with NDEA-induced group (II) rats. 3.4. TQ recovers the hepatic architecture in NDEA induced hepatocarcinogenesis The hepatic sections from control (Group I) rats revealed hepatic lobules with normal liver parenchymal architecture (Fig. 3f), NDEA-administered (Group II) rats showed loss of architecture with pleomorphism of nuclei. The neoplastic hepatocytes exhibits granular cytoplasm with larger hyperchromatic malignant nuclei (HCM), and the tumor cells containing cytoplasmic vacuoles (V) with cellular infiltration (Fig. 3g). Architecture of liver section in rats with TQ administration (Group III), exhibited similar architecture like control (Group I) rats indicating the non-toxic nature of TQ (Fig. 3h), whereas TQ + NDEA administered (Group IV) pre-treated rats liver sections showed normal architecture with an isokaryosis minimal inflammatory and few neoplastically transformed cells of hepatocytes (Fig. 3i), the NDEA + TQ administered (Group V) rats improved hepatocellular characteristics, i.e. more regular and less altered hepatocytes (Fig. 3j) compared with NDEA-administered (Group II) rats were observed. 3.5. Anti-proliferative effect of TQ as revealed by AgNOR staining Fig. 3k–o and p shows the effect of TQ on the histochemical analysis of Argyrophilic Nucleolar Organizing Regions (AgNORs) staining in control and experimental rats. The NDEA-administered liver tumor bearing (Group II) rats showed significantly (p < 0.05) increased number of the AgNORs dots/nuclei when compared with control (Group I) rats. Though, the TQ pre- and post-treated (Groups IV and Group V) rats area of AgNORs dots/nuclei levels were significantly (p < 0.05) decreased when compared with tumor bearing NDEA-administered (Group II) rats. This result clearly exhibits antiproliferative activity of TQ, based on inhibition or arrest of some degree of cancer cell proliferation in both neoplastic lesion and tissues surrounding tumors. 3.6. TQ decreases the levels of liver tumor markers

3.3. TQ diminish the levels of liver injury markers Table 4 depicts the effects of TQ on the activities of liver marker enzymes AST, ALT, ACP, ALP, LDH, GGT, 5 -NT, and total bilirubin levels in the serum of control and experimental groups of rats. The NDEA-administered (Group II) rats reflects significant (p < 0.05) elevation in the liver cell metabolism, which leads to distinctive changes in liver marker enzymes when compared with normal control (Group I) rats; increased enzymes levels are indicators of liver

Fig. 4 describes the effect of TQ on the levels of AFP and CEA, which are the most extensive tumor markers used in the diagnosis of HCC. Therefore, high levels of AFP and CEA are believed to be a strong indicator of hepatic carcinogenesis because more than 80% of HCC patients have high serum concentration of AFP and CEA due to tumor burden. In NDEA-administered (Group II) rats, there was a significant (p < 0.05) increase in the levels of these tumor markers compared with normal control (Group I) rats, which could be due to

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Fig. 3. Effect of thymoquinone on macroscopic appearance of rat liver tumors (a–e); representative micrographs of hematoxylin and eosin (H and E) staining (f–j); argyrophilic nucleolar organizer regions (AgNORs) staining (k–o) in rat liver of control and experimental group of rats. Macroscopically visible hepatic nodules are depicted by red arrows. Representative livers were excised from experimental groups 18 weeks following the initiation of the study: (a) shows the liver from normal control rat (Group I) showing normal surface and absence of nodules; (b) shows the NDEA administered rat (Group II) showing abnormal appearance with a large nodule in many lobes; (c) shows the liver from drug control rat (Group III – TQ alone) showing natural appearance like normal control liver; (d) shows liver from pretreated rat (Group IV) shows customary appearance with a small; (e) shows liver from posttreated rat (Group V) shows general appearance with significantly reduced incidence of tumor nodules and multiplicity. Representative micrographs of histologic sections were shown at 20× magnification. To evaluate cellular damage, sections of control and experimental groups were evaluated by three independent and blinded observers. (f) Showing normal hepatic cells with well preserved/granulated cytoplasm; brought out central vein; prominent nucleus and nucleolus from a normal (Group I) rats. (g) Showing loss of architecture, a marked tendency to spread by intrahepatic veins, many of the tumor cells contain intracytoplasmic violaceous, hyaline globules (arrow) that represent proteins produced by the tumor cells from NDEA (0.01%) administered (Group II) rats. (h) Showing normal liver architecture from TQ alone (Group III) rats. (i) Showing few neoplastic hepatocytes and normal hepatic lobule architecture from TQ + NDEA pretreated (groups IV) rats. (j) Showing loss of architecture with some hepatocytes showing an isokaryosis minimal inflammatory cell infiltration around the portal triads, comparatively less tendency to spread by intrahepatic veins, both hepatic and portal vessels from a NDEA + TQ post treated (Group V) rat. (k) Shows normal architecture with minimal nuclear stain of AgNORs in the normal control (Group I) rats, (l) shows increased nuclear stain of AgNORs in NDEA-induced HCC bearing (Group II) rats, (m) TQ alone administered (Group III) rats, (n and o) Shows reduced nuclear stain expression of AgNORs positive cell in TQ treated (pre and post-treated) Groups IV and V rats. Arrow indicates the nuclear stain expression of AgNOR positive cell. (p) The representative graphs show the percentage of area of AgNORs stain in control and experimental group of rats. AgNORs stain was assessed and measured objectively by two independent observers and gave similar results. For quantitation, data expressing the respective nuclear stain of AgNORs were quantified by counting the positively stained cells in ten fields/section by three independent observers in blinded fashion, and the average was used to denote the total no. of positively stained cells, that was presented as “fold changes” as compared with control and experimental group of rats. Results are expressed as mean ± SD (n = 10), p < 0.05, compared with a Group I, b Group II, c Group III.

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Fig. 3. (continued ).

tumor cell proliferation. However, the levels of these tumor markers were significantly (p < 0.05) abridged in TQ Pre and Post-treated (Group IV and Group V) rats, which was presumably due to inhibition of malignant transformation and therefore decreases in the tumor production rates that are associated with anti-proliferative effect of TQ.

3.7. Immunofluorescence, immunoblot and RT-PCR analysis of cell proliferative markers Fig. 5a–c shows the confocal immunofluorescence, immunoblotting and RT-PCR analysis to confirm the protein and mRNA expression levels of cell proliferative markers (PCNA and Ki-67) in control and experimental rats. The NDEA administered (Group II) rats reveal the protein and mRNA expression levels of PCNA and Ki-67 was significantly (p < 0.05) high as compared to the control (Group I) rats. Whereas, the TQ pre- and post-treated

Fig. 4. The representative graphs shows the Levels of tumor markers AFP and CEA in the serum of control and experimental group of rats. Each value is expressed as mean ± SD (n = 10), p < 0.05, compared with a Group I, b Group II and c Group V. Units: ng/ml for AFP and CEA.

Table 3 Effects of thymoquinone treatment on the development and growth of macroscopic hepatocyte nodules induced by NDEA in rats. Groups (n = 10)

II – (NDEA) IV – (TQ + NDEA) V – (NDEA + TQ)

Rats with nodules/total rats

10/10 5/10 8/10

Nodule incidence (%)

100 55 81

Total no. of nodules

Average number of nodules/nodule

224 48 135

23.8 ± 4.8 9.4 ± 2.1a , c 16.2 ± 3.8a , b b,c

Nodules relative to size (% of total no)

≥3 mm

<3 to >1 mm

≤1 mm

47 ± 7 36 ± 4 42 ± 6

29 ± 5 26 ± 3 33 ± 4

27 ± 5 32 ± 4 32 ± 4

Results are expressed as mean ± SD (n = 10). a Statistical significance at p < 0.05 compared with Group II. b Statistical significance at p < 0.05 compared with Group IV. c Statistical significance at p < 0.05 compared with Group V. Table 4 Effects of thymoquinone on the activities of marker enzymes in the tissue of control and experimental groups of rats. Particulars

Group I Control

AST ALT ALP LDH ␥-GT 5 -NT TBL (mg/100 ml)

128.72 48.68 52.36 76.81 18.34 85.71 0.86

Group II NDEA-induced ± ± ± ± ± ± ±

10.9 4.01 4.8 5.9 1.9 6.3 0.04

384.68 144.47 108.96 187.73 34.56 196.24 1.64

± ± ± ± ± ± ±

23.8a 13.1a 12.5a 12.5a 3.01a 12.3a 0.12a

Group III Drug control (TQ alone) 131.56 49.73 53.93 78.29 20.18 88.24 0.91

± ± ± ± ± ± ±

11.4 4.1 5.2 6.1 1.4 6.8 0.05

Group IV Pre treatment (TQ + NDEA) 186.31 78.23 72.62 98.38 26.67 124.65 1.14

± ± ± ± ± ± ±

15.2b , c 6.3b , c 7.6b , c 8.7b , c 1.9b , c 9.7b , c 0.08b , c

Group V Post treatment (NDEA + TQ) 254.69 114.98 92.46 139.47 30.09 176.19 1.49

± ± ± ± ± ± ±

20.3b 11.3b 10.9b 11b 2.7b 11.6b 0.12b

Each value is expressed as mean ± SD (n = 10). a Statistical significance at p < 0.05 compared with Group I. b Statistical significance at p < 0.05 compared with Group II. c Statistical significance at p < 0.05 compared with Group V. Units: ␮moles of pyruvate liberated mg protein per min for AST, ALT and LDH; ␮moles of phenol liberated mg protein per min ALP; nmoles of p-nitroaniline formed mg protein per min for GGT; nmoles of Pi liberated mg protein per min for 5 -NT.

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Fig. 5. (a) Representative micrographs of immunofluorescence expression of PCNA in control and experimental groups of rats. The tissue sections were immunostained with anti-PCNA antibody and an FITC-conjugated secondary antibody (green) and it counterstained with PI (red). Excitation wavelength/emission wavelength of 529 nm/620 nm for PI and 494 nm/525 nm for FITC. Cells were visualized under confocal microscope (Leica TCS-SP2 XL) (scale bar = 50 ␮m). (b) Representative micrographs of immunofluorescence expression of Ki-67 in control and experimental groups of rats. The tissue sections were immunostained with anti-Ki-67 antibody and an FITC-conjugated secondary antibody (green) and it counterstained with PI (red). Excitation wavelength/Emission wavelength of 529 nm/620 nm for PI and 494 nm/525 nm for FITC. Cells were visualized under confocal microscope (Leica TCS-SP2 XL) (scale bar = 50 ␮m). (c) Effect of thymoquinone alimentation on PCNA and Ki-67 immunoblot, RT-PCR and densitometric analysis of control and experimental groups of rats. (A and C) Representative Western blots analysis of PCNA and Ki-67. Protein samples (50 ␮g/lane) resolved on SDS PAGE analysis. ␤-Actin was used as loading control. (B and D) Representative Reverse Transcriptase-PCR analysis. 5 ␮g of total RNA was reversed transcribed and 100 ng cDNA was amplified by PCR using specific primer sets. ␤-Actin was used as an internal control. Quantitative data expressing the corresponding protein and mRNA levels was assessed using densitometry and is expressed in relative intensity as compared with control. The mean mRNA and protein expression from experimental groups were designated as 100% in the graph. Each values represents mean ± SD for n = 10 from three separate experiments, p < 0.05 compared with a Group I, b Group II, and c Group V. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 5. (continued )

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Fig. 5. (continued ).

(Group IV and Group V) animals exhibited a significant (p < 0.05) reduction in the levels of these cell proliferative markers when compared with NDEA administered (Group II) rats. The TQ alone treated (Group III) rats showed expression similar to control (Group I) rats. From these results, it was clear that TQ could effectively inhibit the cell proliferation during HCC. 3.8. Prognostic significance of Cell-Cycle Regulatory Protein expression by Western blot analysis Fig. 6 represents the immunoblotting and densitometric analysis to confirm the protein expression levels of p21WAF1/CIP1 , Cyclin D1, Cyclin E and CDK4 in the control and experimental rats. In NDEA

administered (Group II) rats exhibits significantly reduced levels of p21WAF1/CIP1 (p < 0.05) and concomitantly the cell cycle regulatory proteins such as Cyclin D1, Cyclin E and CDK4 expression levels were significantly (p < 0.05) increased as compared to the control (Group I) rats. Whereas the TQ pre- and post-treated (Group IV and Group V) rats exhibited a significant (p < 0.05) increase in the levels of p21WAF1/CIP1 and significant (p < 0.05) decreased in the levels of Cyclin D1, Cyclin E and CDK4 expression when compared with NDEA administered (Group II) rats. However TQ alone treated (Group III) rats showed expression similar to control (Group I) animals. From these results shows that TQ treatment regulates the cell cycle thereby suppresses tumor growth by inhibiting the cell proliferation during hepatocellular carcinogenesis.

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Fig. 6. Effect of thymoquinone alimentation on cell cycle regulatory proteins in control and experimental groups of rats. Representative immunoblots of (a) p21WAF1/CIP1 , (b) Cyclin D1, (c) Cyclin E and (d) CDK4 protein expressions in the control and experimental rats. Protein samples (50 ␮g/lane) resolved on SDS PAGE analysis. ␤-Actin was used as loading control. Quantitative data expressing the corresponding protein were assessed using densitometry and is expressed in relative intensity as compared with control. The mean protein expressions from experimental groups were designated as 100% in the graph. Each values represents mean ± SD for n = 10 from three separate experiments, p < 0.05 compared with a Group I, b Group II, and c Group V.

4. Discussion Dysregulated proliferation is one of the main characteristic mechanisms for tumorigenesis which acquires at different stages of cancer development like initiation, promotion and progression of tumor growth, thereby increasing the tumor burden, and initiating the metastasis (Gupta et al., 2010). This dysregulation mainly caused by disrupted G1/S phase cell cycle transition during the development of malignant neoplasm (King and Cidlowski, 1998;

Noh et al., 2011). Therefore, the present study was conducted to evaluate the anti proliferative activity of TQ through modulating the cell cycle regulator proteins during NDEA induced rat HCC. In the present study, the food, water intake and the body weight of control and experimental rats were monitored. The body weight of NDEA-administered rats was significantly lowered and this unintentional weight loss was derived from loss of both muscle mass and fat mass. An increased liver weight in hepatocellularcarcinoma bearing animals indicates an uncontrolled proliferation

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of the cancer cells, thereby increases tumor mass and liver weight, observed as a common symptom of carcinogenesis (Naveenkumar et al., 2012). In contrast, TQ treatment showed significant improvement in body weight with reduced liver weight, which indicating that TQ has suppress the tumor growth during NDEA-induced HCC rats. Moreover, the TQ treatment reduces NDEA-induced hepatic tumorigenesis in rats by inhibiting the development of nodules more than 3 mm in size with a concomitant reduction of nodular volume by TQ is an another conspicuous tracking of this study. The suppression of nodule growth and hastening of nodule regression by TQ as observed in the current study could be viewed as a pivotal step for anti-proliferation and inhibition of tumor growth. Our results were supportive to the recent evidence that TQ is cytotoxic to solid tumors such as colon cancer, pancreatic cancer and forestomach carcinogenesis (Gali-Muhtasib et al., 2008; Banerjee et al., 2009; Badary et al., 1999). Hepatospecific enzymes are one of the most extensively investigated diagnostic markers for identifying the liver damage and it has been used as a prospective biomarker in various liver diseases and HCC (Cizginer et al., 2011). Previous studies also demonstrated that analysis of hepatospecific enzymes such as AST, ALT, ACP, ALP, LDH, GGT, 5 -NT and total bilirubin as the most sensitive and dramatic indicator of liver damage associated with the mortality of various liver diseases (Alkharfy et al., 2011; Whitfield, 2001; Jagan et al., 2008; Jones and Thompson, 2009). In this study, the marker enzymes AST, ALT, ACP, ALP, LDH, GGT 5 -NT and total bilirubin levels exceeded their normal range in NDEA-induced animals’ when compared with the control rats indicating the carcinogenic nature of NDEA (El-Beshbishy et al., 2011), which may be due to metabolic dysfunction of liver and this is mainly due to leakage of these enzymes from liver cytosol into blood (Ramakrishnan et al., 2007). The activity of hepatospecific enzymes levels was restored to near normal level in TQ (pre and post)-treated rats. These results demonstrated that TQ treatment can prevent NDEAinduced hepatic preneoplastic nodule formation and uncontrolled cell proliferation. The observed protection afforded by TQ is probably due to reduced levels of these liver damage markers and total bilirubin levels, thereby inhibiting the dysregulated cell proliferation. The tumor markers such as AFP and CEA are associated with cancer and whose measurement or identification is useful in the diagnosis of cancer and monitoring the prognostic value. AFP is one of the most sensitive and specific marker for diagnosis of HCC, the elevation of AFP occurs only in acute and chronic viral hepatitis as well as in patients with cirrhosis caused by hepatitis C, and it has been linked with proliferation of hepatocyte (Davis et al., 2008). CEA is mainly associated with tumor and used to monitor the recurrence of the diseases. The elevated CEA occurs in the advanced stage of incurable cancer like unresectable HCC. According to Li Gong and Gian Luca Gvazi, the combinational serological analysis of AFP and CEA, are being considered as a most extensively used standard tumor marker in the diagnosis and prognostic determinant value for HCC (Grazi et al., 1995; Gong et al., 2011). In the present study, the AFP and CEA levels were drastically increased in the NDEAadministered rat; which indicates the progression of HCC. Our results suggest that, the liver tumor markers AFP and CEA were significantly lowered the TQ (pre and post)-treated groups when compared with NDEA-induced groups; which clearly states that TQ inhibits the aberrant tumor production and metastasis, resulting in the inhibition of uncontrolled cell proliferation in tumor cells. Cell proliferation plays a crucial role for immortality and is closely related to the promotion of multistage levels of the carcinogenesis. Argyophillic nucleolar organizing regions (AgNORs) are the sites of ribosomal RNA, increased number of AgNORs might reflect an increased rDNA transcription, which might indicate increased nucleolar and cellular activities. Therefore, counting of AgNORs

stain has correlate with cell proliferation and grade of malignancy (Asokkumar et al., 2012). In particular, Kagawa et al. (2004), reported that AgNORs scores for HCC were significantly higher than non-malignant tissue. In our observation, we found that the hyper AgNORs expression as well as defined block dots/nuclei was inhibited by treatment with TQ against NDEA-induced hepatocellular carcinoma, which reflects that treatment with TQ reducing the tumor cell proliferation. The anti-proliferative action of TQ was further confirmed by using cell proliferative markers PCNA and Ki67. PCNA (cell cycle related protein) also known as cyclin, synthesized in early G1 to S-phase function in the cell cycle progression, DNA replication and DNA repair (Huawei et al., 2003), and Ki67 (nuclear protein) expressed in cell proliferation may be required for maintaining cell proliferation and it has been used to evaluate the solid tumor cell proliferation, TQ abrogated the progression of prostate cancer cells from the G1 to S phase cell cycle arrest (El-Mahdy et al., 2005). Here we observed increased expression of PCNA and Ki67 in NDEA exposed liver cells which indicate the involvement of PCNA and Ki67 in higher cell proliferation during the tumorigenesis. Concomitantly, these proliferative markers’ expression was efficiently decreased in TQ (pre and post) treatment indicating the inhibition of cancer cell proliferation against NDEA induced tumorigenesis. These findings prove that TQ can inhibit the cell proliferation and malignant transformation from tumor cells through G1/S phase cell cycle arrest, thereby inhibit the HCC. Major cell cycle regulatory proteins such as cyclins D1, E and CDK4 complexes are essential and mainly involved in the G1/S phase transition during cell cycle in normal cells. Over expression of these cyclin altered the cell cycle progression which is closely associated with malignancy. The dysregulated cell proliferation plays an important role in multistage oncogenesis, which is mainly due to over expression of cyclins D1, E, and CDK4 complexes (Gupta et al., 2010; Lapenna and Giordano, 2009). Many studies suggested that uncontrolled cell proliferation evident from over expression of Cyclin D1, E and CDK4 protein levels are closely associated with variety of human cancer (Gali-Muhtasib et al., 2004; Gupta et al., 2010; Noh et al., 2011), particularly in HCC (Park et al., 2009). Therefore inhibition of the expression of cyclins-CDKs complexes may block the development of carcinogenesis by arresting the G1/S phase dysregulated cell cycle progression. The over-expressed PCNA and Ki67 by alteration of CDK/cyclin family proteins levels which increased DNA synthesis resulting in unchecked cell proliferation and tumorigenesis (Kuwano et al., 1998; Gyoon et al., 2001). In addition, dysregulation of p21WAF1/CIP1 expression and over expression of G1/S phase cell cycle proteins levels are commonly observed in a variety of human cancer (Lapenna and Giordano, 2009; Noh et al., 2011). In this respect, the NDEA administered (Group II) rats can cause uncontrolled cell proliferation which explore the tumorigenic function of NDEA by accelerating abnormal cell cycle progression in HCC. Our results demonstrated that TQ treated (Group IV and V) rats selectively induced p21WAF1/CIP1 , thereby repress the CDK4/cyclin complex expression in G1/S transition suggests a potent anti proliferative role of TQ in the cell cycle arrest during HCC. Our results are congruent with previous studies which have shown that TQ regulates the cell cycle progression via downregulation of cell cycle regulatory proteins, including Cyclin D1, Cyclin E, and cyclindependent kinases (CDK4) with an increase of p2WAF1/CIP1 , thereby inhibiting the cancer cell proliferation. In conclusion, the data provides strong evidence that inhibition of cell proliferation in neoplastic hepatocytes and the surrounding areas of hepatic tissue by TQ is a major mechanism that may be closely related to inhibition of hepatocarcinogenesis. This inhibition might lead to suppression of the hepatospecific marker enzymes, and tumor invasion. Furthermore, it is possible that the inhibitory efficacy of TQ on dysregulated cell proliferation and

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G1/S phase cell cycle arrest may act at mid stage of hepatocarcinogenesis. Hence, TQ can be used as an adjunct to conventional chemopreventive agent, which may provides a novel therapeutic approach to serve as promising agent for treatment of hepatocellular carcinoma. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments This work is supported in part by a fund from Indian Council for Medical Research (ICMR), Government of India, New Delhi. We thank Dr. Ramamurthy, Director, Ultrafast process laboratory, University of Madras for his help and advice in confocal imaging and Prof. Dr. M. Michael Aruldhas, Department of Endocrinology, University of Madras for his help and advice in Chemi-doc imaging, is gratefully acknowledged by the first author. References Alkharfy, K.M., Al-Daghri, N.M., Al-Attas, O.S., Alokail, M.S., 2011. The protective effect of thymoquinone against sepsis syndrome morbidity and mortality in mice. Int. Immunopharmacol. 11, 250–554. Asokkumar, S., Naveenkumar, C., Raghunandhakumar, S., Kamaraj, S., Anandakumar, P., Jagan, S., Devaki, T., 2012. Antiproliferative and antioxidant potential of beta-ionone against benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice. Mol. Cell. Biochem. 363, 335–345. Badary, O.A., Al-Shabanah, O.A., Nagi, M.N., Al-Rikabi, A.C., Elmazar, M.M., 1999. Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone. Eur. J. Cancer Prev. 8, 435–440. Banerjee, S., Kaseb, A.O., Wang, Z., Kong, D., Mohammad, M., Padhye, S., 2009. Antitumor activity of gemcitabine and oxaliplatin is augmented by thymoquinone in pancreatic cancer. Cancer Res. 69, 5575–5583. Brown, J.L., 1999. N-nitrosamines. Occup. Med. 14, 839–848. Cizginer, S., Tatli, S., Hurwitz, S., Tuncali, K., vanSonnenberg, E., Silverman, S.G., 2011. Biochemical and hematologic changes after percutaneous radiofrequency ablation of liver tumors: experience in 83 procedures. J. Vasc. Interv. Radiol. 22, 471–478. Das, S., Dey, K.K., Dey, G., Pal, I., Majumder, A., MaitiChoudhury, S., kundu, S.C., Mandal, M., 2012. Antineoplastic and apoptotic potential of traditional medicines thymoquinone and diosgenin in squamous cell carcinoma. PLoS One 7, 10. Davis, G.L., Dempster, J., Meler, J.D., Orr, D.W., Walberg, M.W., Brown, B., 2008. Hepatocellular carcinoma: management of an increasingly common problem. Proc. Bayl. Univ. Med. Cent. 21, 266–280. El-Beshbishy, H.A., Tork, O.M., El-Bab, M.F., Autifi, M., 2011. Antioxidant and antiapoptotic effects of green tea polyphenols against azathioprine-induced liver injury in rats. Pathophysiology 18, 125–135. El-Mahdy, M., Zhu, Q., Wang, Q.E., Wani, G.A., 2005. Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells. Int. J. Cancer 117, 409–417. Gali-Muhtasib, H., Ocker, M., Kuester, D., Krueger, S., El-Hajj, Z., Diestel, A., 2008. Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models. J. Cell. Mol. Med. 12, 330–342. Gali-Muhtasib, H., Roessner, A., Schneider-Stock, R., 2006. Thymoquinone: a promising anti-cancer drug from natural sources. Int. J. Biochem. Cell Biol. 38, 1249–1253. Gali-Muhtasib, H.U., Abou Kheir, W.G., Kheir, L., Darwiche, N., Crooks, P., 2004. Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes. Anticancer Drugs 15, 389–399. Gong, L., Zheng, M., Li, Y., Zhang, W., Bu, W., Shi, L., 2011. Seminal vesicle metastasis after partial hepatectomy for hepatocellular carcinoma. BMC Cancer 11, 111. Grazi, G.L., Mazziotti, A., Legnani, C., Jovine, E., Miniero, R., Gallucci., 1995. The role of tumor markers in the diagnosis of hepatocellular carcinoma, with special reference to the des-gamma-carboxy prothrombin. Liver Transplant. Surg. 1, 249–255. Gupta, S.C., Kim, J.H., Prasad, S., Aggarwal, B.B., 2010. Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation ofinflammatory pathways by nutraceuticals. Cancer Metastasis Rev. 29, 405–434. Gurung, R.L., Lim, S.N., Khaw, A.K., Soon, J.F., Shenoy, K., Mohamed Ali, S., Jayapal, M., Sethu, S., Baskar, R., Hande, M.P., 2010. Thymoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. PLoS One 5 (8). Gyoon, C., Min Gyu, K., Won Sang, P., Jung Young, L., Suk Woo, N., Dong, Y., Sui, L., Sugimoto, K., Tai, Y., Tokuda, M., 2001. Cyclin D1–CDK4 complex, a possible critical factor for cell proliferation and prognosis in laryngeal squamous cell carcinomas. Int. J. Cancer 95, 209–215.

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