Functional Evaluation of Imatinib mesylate in ...

Send Orders of Reprints at [email protected] Recent Patents on Biomarkers 2013, 3, 000-000

1

Functional Evaluation of Imatinib mesylate in Hepatocellular Carcinoma Cells Mai A. SaadZaghloul1, Ashraf H. Abadi2 and Ahmed I. Abdelaziz1,* 1

The Molecular Pathology Research Group, Department of Pharmacology and Toxicology, Faculty of Pharmacy and Biotechnology, German University in Cairo, Egypt; 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Egypt. Received: August 29, 2012; Accepted: October 10, 2012; Revised: November 06, 2012

Abstract: Hepatocellular Carcinoma remains a major fatal disease that is resistant to most cytotoxic therapeutics owed to the aberrant activation of signalling cascades. Recent patents reveal a new family of drugs that has been studied for molecularly targeting these cascades; multi-kinase inhibitors, are nowadays considered as novel therapeutic approaches. Therefore in this study we aimed at investigating the impact of Imatinib mesylate, a tyrosine kinase inhibitor, on Human Hepatoma (HuH-7) cellular behavior and specifically its effect on p53 tumor suppressor gene. HuH-7 cells were transfected with a reporter vector containing a specific enhancer element that is activated upon binding to intracellular p53; consequently downstream luciferase reporter gene is activated. Cells were treated with Imatinib and we looked for p53 induction upon drug stimulation. Additionally; viability, metabolism, proliferation and apoptosis were evaluated. Upon Imatinib treatment, p53 expression showed a significant increase represented in increased luminescence. Moreover; a decrease in cellular viability and metabolic activity along with a considerable inhibition of proliferation and a vast increase in Caspase 9 activity were observed when compared to untreated cells. This study suggests that the effects of Imatinib mesylate might be attributable to enhanced active p53 in HuH-7 cells with consequent reduction in cancer progression properties. The article also summarizes some recent relevant patents.

Keywords: Hepatocellular carcinoma, HuH-7, imatinib mesylate, luciferase reporter gene, molecular targeted therapy, p53. INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most aggressive cancers worldwide; where over 600 000 deaths occur annually due to this disease [1]. Although several HCC treatments are available, yet they are restricted to the stage at which the cancer is diagnosed. The majority of HCC patients are usually diagnosed at a very late stage which makes the potentially curative therapy the least effective. While most of the early diagnosed patients are limited to either liver transplant or resection and only 10-15% are suitable candidates for medical treatments to which they become resistant shortly after [2]. HCC is considered a heterogeneous tumor with several genomic alterations causing aberrant activation of signaling pathways such as Ras/Raf/Mek [3], PI3k/Akt [4], and in addition to several signaling molecules such as p53 tumor suppressor protein, consequently halting its function in inhibiting proliferation and inducing apoptosis [5]. This creates a research challenge to fulfil an urging need for new therapeutic strategies that could be curative for the majority of patients with this aggressive disease. A recently emerging class of therapeutics is now focusing on targeting specific signaling molecules contributing to the pathogenesis *Address correspondence to this author at the Molecular Pathology Research Group, Department of Pharmacology and Toxicology, Faculty of Pharmacy and Biotechnology, The German University in Cairo (GUC), New Cairo City - Main Entrance Al Tagamoa Al Khames 11835, Cairo, Egypt; Tel: +20-2-27590714; Fax: +20-2-27581041; E-mail: [email protected]

2210-3090/13 $100.00+.00

of HCC tumors in an attempt to reverse the progress of the disease and diminish the resistance to therapy; one example is the multi-kinase inhibitors. Sorafenib tosylate (Nexavar® tablets, Bayer Pharmaceuticals Corp.), a small molecule Raf kinase inhibitor and Vascular Endothelial Growth Factor (VEGF) receptor kinase inhibitor, has been approved in 2007 by the Food and Drug Administration (FDA) for the treatment of patients with unresectable hepatocellular carcinoma (HCC) as the first systemic therapeutic approach [6]. Sorafenib has shown to increase overall survival and time to progression in advanced unresectable HCC patients [7] and decrease the development of post surgical intrahepatic recurrences and abdominal metastasis in mice models [8]. Although the direct action of Sorafenib on p53 is not elucidated, it has been reported that its action on Mitogenactivated Protein Kinase (MAPK) signaling is differential according to the status of p53 [9]. Additionally, Imatinibmesylate (STI-571; Gleevec® tablets, Novartis Pharmaceuticals Corp.); a tyrosine kinase inhibitor, targets specifically bcr/abl [10], PDGF-R [11] and ckit [12], is indicated for the treatment of Philadelphia chromosome-positive (Ph+) Chronic Myelogeneous Leukemia (CML) [10] and is approved by the FDA early 2012 as an adjuvant treatment for Gastrointestinal Stromal Tumors (GIST) [13]. However, due to the presence of abrupt mutations and therefore abnormally controlled signaling pathways, resistance to therapy is recurrent. In a recent patency, new means were invented to circumvent the resistance to Imatinib treatment due to Kit mutation in Gastrointestinal © 2013 Bentham Science Publishers

2

Recent Patents on Biomarkers 2013, Vol. 3, No. 1

Tumors (GIST) [14]. Similarly in Chronic Myelogeneous Leukemia (CML), another patency reveals the presence of novel genes and their encoded proteins called Mutants Associated with Resistance to STI-571 (MARS), such as mutated Bcr-Abl complex, this invention also provides means for restraining the functional behavior of the MARS genes and proteins [15]. Conversely, Imatinib mesylate has not been studied in depth for the treatment of HCC specifically its effect on molecular targets contributing to the pathogenesis of the disease [16, 17]. Since Imatinib has shown promising results in studies conducted on GIST patients, revealing its up-regulating effect on p53 tumor suppressor gene [18], it became our interest in this study to have a preliminary evaluation of the effect of Imatinib mesylate on p53 tumor suppressor gene expression in HCC cell model and to assess its effect on the cellular activities of the Human Hepatoma cell line (HuH-7). MATERIAL AND METHODS Cell Lines HuH-7 cell line, harbouring mutant p53 tumor suppressor gene with a point mutation at codon 220 (A:T to G:C) resulted in the amino acid changes of cysteine for tyrosine [19] and HepG2 cell line, harbouring wild type p53 [20]. Cultured in Dulbecco’s Modiﬁed Eagle's Medium (DMEM) supplemented with 4.5g/L Glucose, L-Glutamine and enriched with 10% FBS and 1% penicillin/streptomycin (LONZA, Germany) in an incubator environment of 5% CO2 at 37ºC. Drug Preparation A stock of Imatinib mesylate (Gleevec®; Novartis, Switzerland) was prepared at a concentration of 500µM dissolved in Dimethyl sulfoxide (DMSO) that constitutes 2% in DMEM medium. Control experiments were subjected to the same culturing medium containing 2% DMSO in DMEM medium. Cell Model Preparation A p53 reporter vector was used; containing a specific cisacting DNA sequence (enhancer element) and a luciferase reporter gene that increase luminescence upon binding of the transcription factor p53 to the enhancer element (Pp53-TALuc; Clontech Laboratories, USA). As a negative control vector, pTAL-Luc (Clontech Laboratories, USA) was used to determine un-induced background signals associated with luciferase reporter gene activity. This vector lacks the enhancer element, but contains a promoter and luciferase reporter gene. The experimental values were normalized to the values obtained with the control vectors. Cell Transfection The constructs were transfected in HuH-7 cell line using the Superfect® transfection reagent (Qiagen, Germany) according to the manufacturer’s recommendations. Drug Stimulation A working concentration of 50µM Imatinib mesylate was added to transfected cells and incubated for 48 hours prior reporter gene expression assay.

SaadZaghloul et al.

Luciferase Reporter Gene Expression Assay Prior to the assay, transfected cells were washed, lysed and collected. Centrifuged at 12,000 xg, 4˚C for 1 minute, the supernatant containing the luciferase protein is mixed with the luciferin substrate (Sigma-aldrich, Germany) and luminescence is measured at an integration time of 1 second, according to the manufacturer’s recommendations. Cytotoxicity Assay A range of working concentrations of Imatinib mesylate was tested after 24 and 48 hours of drug exposure to HuH-7 cells prepared at the log-phase. MTT assay was carried out according to the manufacturer’s recommendations (Sigmaaldrich, Germany) where the absorbance was measured at 572 nm. Values were obtained for triplicate experiments, used as percentage and normalized to untreated cells. Cell Viability Assay A working concentration of 100µM Imatinib mesylate was used to assess cell viability over several time intervals of treated HuH-7 cells. 0.33% Neutral red solution (Sigmaaldrich, Germany) was added to the cells in an amount equal to 10% of the culture medium volume. The cells were incubated for 3 hours under normal conditions then washed. For cell lysis, a mixture of 1:1:0.02 (distilled water: absolute ethanol: acetic acid) was added in an amount equal to the original volume of culture medium and allowed to stand for 30 minutes at room temperature under gentle shaking. Absorbance was then measured at 572 nm which corresponds to the relative number of viable cells and compared to untreated cells. Values were obtained for triplicate experiments and averaged. Functional Integrity Assay (ATP quantification) The experiment was based upon a bio-luminescent measurement of intracellular ATP present in metabolically active cells whereby an ATP-dependant D-Luciferin oxidation reaction takes place in presence of a Luciferase enzyme, accompanied by emission of light that linearly corresponds to the ATP concentration (ViaLight Plus®; LONZA, Germany). Treated HuH-7 cells with 50 µM Imatinib mesylate for 48 hours of exposure were assessed. Treated HepG2 cells were also assessed at the same conditions (Data not shown). Values were obtained for triplicate experiments and averaged. Proliferation Assay Brdu® Assay (Roche, Germany) was carried out according to the manufacturer’s recommendations, whereby treated HuH-7 cells with 50µM Imatinib mesylate, 48 hours of drug exposure were assayed for proliferation rates and compared to untreated cells. Values were obtained for triplicate experiments and averaged. Cell Apoptotic Assay HuH-7 cells were exposed to 50µM Imatinib mesylate for 48 hours, assayed for the amount of active Caspase 9 and compared to untreated cells. This assay (Caspase-Glo® 9; Promega, Germany) was based on a luminogenic substrate

Hepatocellular Carcinoma Therapy

that upon cleavage by active Caspase 9 yields a luminescent signal that corresponds in value to the level of intracellular active Caspase 9. Values were obtained for triplicate experiments and averaged. Statistical Analysis All data were expressed as the mean ± standard error of the mean (SEM), calculated using unpaired Student’s T-Test to compare between treated and untreated cell lines. A “P” value less than 0.05 was considered statistically significant; * < 0.05, **< 0.01, ***< 0.001. Calculations were performed using the GraphPad® Prism 5.0 Software. RESULTS Preliminary Evaluation of the p53 Protein Level Drug stimulation, of the p53 reporter vector transfected cells, with 50µM Imatinib mesylate have resulted in Eightfolds upregulation of endogenous p53 protein in HuH-7 cell line (32720 + 2822 N = 3) when compared to values obtained from untreated transfected cells that corresponds to p53 basal protein levels (3778 + 834.0 N = 3) (P value = 0.0006). The experimental values were normalized to the values obtained from cells transfected with the control vector in order to eliminate background activity of the luciferase protein Fig. (1).


3

(P value = 0.0039). The values shown represent percentage viable cells relative to untreated cells Fig. (2). Cell Viability Assay HuH-7 cells treated with 100µM Imatinib mesylate for 24 hours showed the optimum decrease in cell viability (0.1027 + 0.002333 N = 3) relative to untreated cells (0.1397 + 0.002333 N = 3) (P value = 0.0004). Which further decreased after 48 hours of drug exposure (0.0940 + 0.003786 N = 3) compared to untreated cells (0.1160 + 0.003215 N = 3) (P value = 0.0114) while a non-significant decrease in cell viability is observed after 72 hours of drug exposure (0.09667 + 0.002028 N = 3) relative to untreated cells (0.1010 + 0.005132 N = 3) (P value = 0.4762). This reveals a possible time-dependant loss of Imatinib mesylate activity as depicted by decreased significant inhibition of cellular viability over time Fig. (3). Functional Integrity Assay (ATP quantification) Treated HuH-7 cells showed a highly significant decrease in ATP levels (795.3 + 105.4 N = 3) when compared to untreated cells (13630 + 2093 N = 3) (P value = 0.0036) which reflects reduced cellular functional and metabolic activity upon drug treatment Fig. (4). Proliferation Assay Treated HuH-7 cells revealed a significant antiproliferative action of Imatinib mesylate (667100 + 71450 N = 3) when compared to untreated cells (837900 + 7296 N = 3) (P value = 0.031) Fig. (5). Cell Apoptotic Assay We measured the level of Caspase 9 enzymatic activity upon drug treatment, which reflects the possible participation of p53 as a key initiator of the intrinsic apoptotic pathway [21]. Caspase 9 enzymatic activity was shown to be highly promoted in HuH-7 cell line upon drug exposure (330300 + 21070 N = 3) relative to untreated cells (69520 + 20020 N = 3) (P value = 0.0009) Fig. (6). DISCUSSION

Fig. (1). Preliminary evaluation of the p53 protein level A comparison between Imatinib-treated Pp53-TA-Luc transfected HuH-7 cells (32720 + 2822 N = 3) and untreated Pp53-TA-Luc transfected HuH-7 cells (3778 + 834.0 N = 3) showed a significant upregulation of endogenous p53 protein (P value = 0.0006). Results were calculated using the unpaired student T-Test and expressed as mean ± standard error of the mean (SEM). Statistical significance is demonstrated as Asterisks (*) representing a significant difference with P value < 0.05.

Cytotoxicity Assay A range of doubling concentrations of Imatinib mesylate was studied for cytotoxicity whereby a dose of 80 µM Imatinib mesylate was lethal to about 25% of the total cell density after 48 hours of exposure (76.49% + 3.891 N = 3)

Deregulation of apoptosis and enhanced cellular proliferation attribute to the development, metastasis, tumor invasion and progression of hepatocellular carcinoma [5]. Disruption in the cell signaling pathways are markedly observed in advanced HCC leading to the disease progression and resistance to therapy therefore limiting the treatment options. Consequently, molecular targeting of cell signalling genes and proteins has become a new demand for treating HCC [22]. P53 tumor suppressor gene is one of the key regulators of cellular proliferation and apoptosis [23, 24]; therefore it is considered a significant target for therapy since several mutations of the p53 gene are observed in HCC, promoting the disease progression and resistance to therapy [25-27]. Targeting the abnormally functioning mitogenic pathways such as the hyperactive receptor and cytoplasmic tyrosine kinases became important targets for therapy in an approach to suppress proliferation [3, 4]. Several studies have investigated the effect of Imatinib mesylate on multiple intracellular mo-

4


SaadZaghloul et al.

Fig. (2). Cytotoxicity Assay. A comparison between Imatinib-treated HuH-7 cell line along a range of doubling concentrations (10, 20, 40, 80, 160µM) after 24 and 48 hours with untreated cells; where at 80µM after 48 hours of drug exposure (76.49 + 3.891 N = 3) a significant cytotoxic effect is shown (P value = 0.0039) while at 160 µM after 24 and 48 hours, a highly significant cytotoxic effect is observed (P value < 0.0001) (56.61 + 2.670 N=3) and (P value = 0.0001) (55.19 + 2.834 N = 3) respectively. Results are shown as % normalized to untreated cells and are calculated using the unpaired student T-Test and expressed as mean ± standard error of the mean (SEM). Levels of statistical significance are demonstrated as Asterisks (*) representing a significant difference with P value < 0.05.

Fig. (3). Cell Viability Assay. A comparison between Imatinib-treated HuH-7 cell line at 100 µM after 24, 48 and 72 hours (0.1027 ± 0.002333 N=3), (0.0940 + 0.003786 N = 3) and (0.09667 + 0.002028 N = 3) respectively and untreated cells (0.1397 + 0.002333 N=3), (0.1160 ± 0.003215 N = 3) and (0.1010 + 0.005132 N = 3) respectively; showing a significant decrease of viability at 24 and 48 hours (P value = 0.0004) and (P value = 0.0114) respectively. While after 72 hours, results showed a non-significant decrease of cell viability (P value = 0.4762). Results were calculated using the unpaired student T-Test and expressed as mean + standard error of the mean (SEM). Levels of statistical significance are demonstrated as Asterisks (*) representing a significant difference with P value < 0.05.

lecular targets in CML; where Liu et al. have revealed a new Imatinib-induced p53-independent pro-apoptotic mechanism through which Imatinib upregulates p38/MAPK, Checkpoint kinase 2 (chk2), p73 and Bax [28]. Another study have demonstrated the correlation between the status of p53 and the response to Imatinib in CML patients; An enhanced induction of apoptosis by Imatinib and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53 [29]. On the contrary, the use of Imatinib mesylate in the molecular targeting of intracellular signaling proteins in hepatocellular carcinoma cells have not been fully investigated to this point specifically targeting the p53 tumor suppressor gene. Therefore this study aimed at inves-

tigating the effect of Imatinib mesylate (a tyrosine kinase inhibitor) on p53 protein expression and several functional analyses in Human Hepatoma cell line (HuH-7). For that purpose, a cell model was created; composed of a reporter vector that contains a specific p53 enhancer element and a sensitive reporter gene thus upon binding of endogenous p53 to the enhancer element, screening for the induction of p53 expression is made easy through analysing the expression of the reporter gene. This model facilitates obtaining preliminary evidence concerning the effect of Imatinib mesylate on the activation of endogenous p53 protein in Human Hepatoma cell line (HuH-7). In order to support our results further functional analyses were also performed.

Hepatocellular Carcinoma Therapy

Fig. (4). Functional Integrity Assay (ATP quantification). A comparison between Imatinib-treated HuH-7 cells (795.3 + 105.4 N = 3) and untreated cells (13630 + 2093 N = 3) showing a highly significant decrease of cellular functional integrity (P value = 0.0036). Results were calculated using the unpaired student TTest and expressed as mean ± standard error of the mean (SEM). Statistical significance is demonstrated as Asterisks (*) representing a significant difference with P value < 0.05.

Fig. (5). Proliferation Assay. A comparison between Imatinib-treated HuH-7 cells after 48 hours drug exposure (667100 + 71450 N = 3) and untreated cells (837900 + 7296 N = 3) showing a significant anti-proliferative effect of the compound (P value = 0.031). Results were calculated using the unpaired student T-Test and expressed as mean ± standard error of the mean (SEM). Statistical significance is demonstrated as Asterisks (*) representing a significant difference with P value < 0.05.


5

Fig. (6). Cell Apoptotic Assay A comparison between Imatinib-treated HuH-7 cells after 48 hours of drug exposure (330300 + 21070 N = 3) and untreated cells (69520 + 20020 N=3) showing a highly significant over-activation of caspase 9 (P value = 0.0009). Results were calculated using the unpaired student T-Test and expressed as mean + standard error of the mean (SEM). Statistical significance is demonstrated as Asterisks (*) representing a significant difference with P value < 0.05.

Interestingly, Imatinib showed a significant up regulation of p53 protein relative to untreated transfected cells (P value = 0.0006) Fig. (1) which could be due to enhanced activity and/or enhanced gene expression of p53 protein. This result relates to another study conducted on GIST patients; revealing the up regulation of p53 upon Imatinib treatment [18]. Additionally, our data was further supported by MTT assay where Imatinib mesylate showed a significant increase in cytotoxicity along a wide range of concentrations (P value ≤ 0.0001) Fig. (2) and a significant decrease in cellular viability after 24 hours of drug exposure relative to untreated cells (P value = 0.0004) Fig. (3). At the metabolic level, Imatinib treatment showed a significant decrease in intracellular ATP levels and hence a decrease in the cellular functional integrity of HuH-7 cells (P value = 0.0036) Fig. (4). Testing the impact of Imatinib on proliferation, drug treatment showed a significant decrease in cellular proliferation of HuH-7 cells when compared to untreated counterparts (P value = 0.031) which might be due to probable cell cycle arrest Fig. (5). This result coincides with a study conducted by Campbell et al. showing a decreased cellular proliferation in hepatocellular carcinoma mice models upon Imatinib treatment [30]. Imatinib mesylate was also explored in this study for its effect on apoptosis in HuH-7 cells through assessing Caspase 9 enzymatic activities which is commonly mediated via p53 activation. Imatinib mesylate was shown to promote apoptosis significantly through enhancement of Caspase 9 activity relative to untreated cells (P value = 0.0009) Fig. (6), revealing the involvement of the intrinsic apoptotic pathway indicating possible participation of the p53 protein of the HuH-7 cell line. The results of our study coincides to a great

6


extent with a study conducted in 2012 providing the first evidence that reactivating mutant p53 and modulating MDM2-p53 pathway may be useful in improving the apoptotic and anti-proliferative responses of Imatinib mesylate and other Kit-inhibiting drugs in the treatment of naive GIST, with p53 mutation status being a predictive factor of response [31]. Bearing in mind that HuH-7 cell line is harboring mutant p53 tumor suppressor gene, it is important to mention that the results obtained in our study could be contributed by an active yet mutant p53 protein that is stimulated by Imatinib treatment. In this case, the enhanced luciferase reporter gene activation was due to upregulation or enhanced activity of mutant p53 protein. Moreover, the decreased proliferation and over-activation of Caspase 9 could be due to the upregulation of active mutant p53 protein upon Imatinib treatment when compared to untreated equivalents. In an initial approach to prove this relation, the effect of Imatinib mesylate on the functional integrity and metabolic activity through measuring cellular ATP levels was analysed on two HCC cell lines; HuH-7 and HepG2 cells. Where in HuH-7 cells, harbouring mutant p53 gene, a highly significant decrease in metabolic activity was observed (P value = 0.0036) unlike HepG2 cell line, harbouring wild-type p53 gene, showed no significant effect on the cellular metabolic activity (P value = 0.4963) (Data not shown). This finding could lead to two different explanations; first, the effect of Imatinib mesylate is dependent on the status of the p53 gene which coincides in principle with a previous study by Goldberg et al. [29]. Second, the expression of stem cell factor receptor (C-Kit) is observed in HuH-7 but not HepG2 cell line which might affect the individual response to treatment [32]; that explains the poor effect of Imatinib mesylate on the suppression of cellular metabolic activity of HepG2 cells. Therefore, the exact p53-dependant mechanism of action of Imatinib mesylate in treating hepatocellular carcinoma needs further investigation. CONCLUSION Imatinib mesylate treatment of human hepatoma cells (HuH-7) showed upregulation and activation of p53 tumor suppressor protein with enhanced cytotoxicity. It also showed significant decrease in cellular viability, with suppressed functional integrity and metabolic activity, and reducing cell proliferation. In addition to enhanced apoptosis via increase in Caspase 9 enzymatic activity. CURRENT & FUTURE DEVELOPMENTS At the meantime, Tyrosine Kinase Inhibitors (TKI) are being the major icons for molecular targeted therapy in an attempt to treat one of the most aggressive cancers; Hepatocellular Carcinoma. Several patents have focused on inventions in modulating TKI drugs in order to achieve better therapeutic efficacy along with restricted liability to resistance. One invention relates to several compounds that are able to modulate, inhibit and/or regulate receptor as well as cytoplasmic tyrosine kinases, therefore control the aberrant activation of signaling transduction presented in many tumors. One compound modulates the catalytic activity of Receptor Tyrosine Kinases (RTK) taking part in several tumors such as

SaadZaghloul et al.

EGF, HER2, HER3, HER 4, IR, IGF-1R, IRR, PDGFR α/β, HGF, and C-Kit [33]. In search for a better solution to the recurrent resistances developed upon anti-cancer treatment options, a patency issued in 2012 claims to have invented a pharmaceutical composition suitable for treating a multidrug resistant cell, it acts as an active agent for modulation of anti-apoptotic pathways and multidrug resistant transporter expression and/or function [34]. Although not many patents are available in the subject of HCC treatment with Imatinib mesylate, yet its mechanism in reversing cancer properties is worth exploring on the molecular level. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGMENTS Cell lines used in this study were kindly provided by Prof. Dr. Peter Schirmacher and Prof. Dr. Kai Breuhahn; Institute of Pathology, University of Heidelberg, Germany. REFERENCES [1]

[2] [3]

[4]

[5] [6]

[7]

[8]

[9]

[10] [11]

[12] [13]

World Gastroenterology Organization-Hepatocellular carcinoma (HCC): A global perspective. Available at: http://www.worldgastroenterology.org/hepatocellularcarcinoma.html (Accessed on: October 27, 2012). Ryder S. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut 2003; 52: iii1-8. Caja L, Sancho P, Bertran E, Iglesias-Serret D, Gil J, Fabregat I. Overactivation of the MEK/ERK Pathway in liver tumor cells confers resistance to TGF-β-induced cell death through impairing upregulation of the NADPH oxidase NOX4. Cancer Res 2009; 69: 7595-602. Suzuki A, Hayashida M, Kawano H, Sugimoto K, Nakano T, Shiraki K. Hepatocyte growth factor promotes cell survival from fas-mediated cell death in hepatocellular carcinoma cells via Akt activation and Fas-death-inducing signaling complex suppression. Hepatology 2000; 32: 796-802. Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 2002; 31: 339-346. Nexavar (sorafenib tosylate) tablets, Prescribing Information March 2011, available at: http://www.fda.gov/Safety/MedWatch/SafetyInformation/ucm2336 95.htm (Accessed on: October 12, 2012). Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008; 359: 378-90. Feng YX, Wang T, Deng YZ, Yang P, Li JJ, Guan DX, et al. Sorafenib suppresses postsurgical recurrence and metastasis of hepatocellular carcinoma in an orthotopic mouse model. Hepatology 2011; 53(2): 483-92. Quack C, Rieser E, Braun M, Massen S, Koch AF, Mueller M, et al. Differential effects of multikinase kinase inhibitors sorafenib and U0126 on phosphorylation patterns of hepatocellular carcinomas cells. Z Gastroenterol 2009; 47-P404. Druke, BJ. Perspectives on the development of a molecularly targeted agent. Cancer Cell 2002; 1: 31-6. Dong Y, Jia L, Wang X, Tan X, Xu J, Deng Z, et al. Selective inhibition of PDGFR by imatinib elicits the sustained activation of ERK and downstream receptor signaling in malignant glioma cells. Int J Oncol 2011; 38(2): 555-69. Akin C, Fumo G, Yavuz AS, Lipsky PE, Neckers L, Metcalfe DD. A novel form of mastocytosis associated with a transmembrane ckit mutation and response to imatinib. Blood 2004; 103: 3222-5. Imatinib Mesylate Tablets, available at: http://www.fda.gov/ Drugs/InformationOnDrugs/ApprovedDrugs/ucm289789.htm (Accessed on: October 25, 2012).

Hepatocellular Carcinoma Therapy [14] [15]

[16] [17]

[18]

[19]

[20]

[21] [22] [23]

[24]

Chen, L.L., Frazier, M.L. Mutations in kit confer Imatinib resistance in gastrointestinal stromal tumors. US7329495 (2008). Sawyers, C.L., Gorre, M.E., Shah, N.P., Nicoll, J. Mutations in the BCR-ABL tyrosine kinase associated with resistance to STI-571. US20030158105 (2003). Lin AY, Fisher GA, So S, Tang C, Levitt L. Phase II study of imatinib in unresectable hepatocellular carcinoma. Am J Clin Oncol 2008; 31: 84-8. Ramadori G, Fuezesi L, Grabbe E, Pieler T, Armbrust T. Successful treatment of hepatocellular carcinoma with the tyrosine kinase inhibitor imatinib in a patient with liver cirrhosis. Anticancer Drugs 2004; 15: 405-9. Romeo S, Debiec-Rychter M, Van Glabbeke M, Van Paassen H, Comite P, Van Eijk R, et al. Cell cycle/apoptosis molecule expression correlates with imatinib response in patients with advanced gastrointestinal stromal tumors. Clin Cancer Res 2009; 15: 419-8. Hsu IC, Tokiwa T, Bennett W, Metcalf RA, Welsh JA, Sun T, et al. p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines. Carcinogenesis 1993; 14: 987-92. Bressac B, Galvin KM, Liang TJ, Isselbacher KJ, Wands JR, Ozturk M. Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc Natl Acad Sci USA 1990; 87: 19737. Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: Structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 1999; 68: 383-424. Thomas M. Molecular targeted therapy for hepatocellular carcinoma. J Gastroenterol 2009; 44: 136-41. Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 1996; 379: 88-91. Nikiforov MA, Hagen K, Ossovskaya VS, Connor TM, Lowe SW, Deichman GI, et al. p53 Modulation of anchorage independent growth and experimental metastasis. Oncogene 1996; 13: 1709-19.

Recent Patents on Biomarkers 2013, Vol. 3, No. 1 [25] [26] [27] [28]

[29]

[30]

[31]

[32]

[33] [34]

7

Mirzayans R, Murray D. Pharmacological modulation of p53 function in cancer therapy. Curr Signal Transd T 2008; 3: 183-94. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991; 253: 49-53. Szymanska K, Hainaut P. Tp53 and mutations in human cancer. Acta Biochim Pol 2003; 50: 231-8. Liu JH, Liu CC, Yen CC, Gau JP, Wang WS, Tzeng CH. Pml and TAp73 interacting at nuclear body mediate imatinib-induced p53independent apoptosis of chronic myeloid leukemia cells. Int J Cancer 2009; 125: 71-7. Goldberg Z, Levav Y, Krichevsky S, Fibach E, Haupt Y. Treatment of chronic myeloid leukemia cells with imatinib (STI571) impairs p53 accumulation in response to DNA damage. Cell Cycle 2004; 3: 1188-95. Campbell JS, Johnson MM, Bauer RL, Hudkins KL, Gilbertson DG, Riehle KJ, et al. Targeting stromal cells for the treatment of platelet-derived growth factor C-induced hepatocellular carcinogenesis. Differentiation 2007; 75: 843-852. Henze J, Meuhlenberg T, Simon S, Grabellus F, Rubin B, Taeger G, et al. p53 Modulation as a Therapeutic Strategy in Gastrointestinal Stromal Tumors. PLoS ONE 2012; 7:e37776. doi:10.1371/ journal.pone.0037776 Mansuroglu T, Baumhoer D, Dudas J, Haller F, Cameron S, Lorf T, et al. Expression of stem cell factor receptor c-kit in human nontumoral and tumoral hepatic cells. Eur J Gastroenterol Hepatol 2009; 21: 1206-11. Kim, A.S. Tyrosine Kinase inhibitors. US20060276514 (2006). Toole, B.P., Misra, S., Ghatak, S. Methods and compositions for inhibition of multi-drug resistance by Hyaluronan Oligomers. US8093217 (2012).