Current Pharmaceutical Design

ISSN: 1381-6128

Current Pharmaceutical Design
Volume 13, Number 32, 2007

Contents

Resent and Future Therapies for the Hepatocellular Carcinoma
Executive Editor: G. Giannelli


Editorial Pp. 3264


Liver Transplantation for Hepatocellular Carcinoma: Current Role and Future Opportunities Pp. 3265-3273
F.L. Koller, S.K. Geevarghese and D.L. Gorden
[Abstract]


Transcutaneous Treatments of Hepatocellular Carcinoma in Patients with Cirrhosis: Present Status and Future Developments Pp. 3274-3278
M. Beaugrand and O. Seror
[Abstract]


Molecular Pathways and Related Target Therapies Pp. 3279-3287
S. Tommasi, R. Pinto, B. Pilato and A. Paradiso
[Abstract]


Rationale for New Drugs Targeting the Tissue Microenvironment in Patients with HCC Pp. 3288-3291
S. Antonaci and G. Giannelli
[Abstract]


The Development of Targeted Therapies for Hepatocellular Cancer Pp. 3292-3300
D. Chattopadyhay, D.M. Manas and H.L. Reeves
[Abstract]


Tyrosine Kinase Inhibitors: A Potential Approach to the Treatment of Hepatocellular Carcinoma Pp. 3301-3304
G. Giannelli, N. Napoli and S. Antonaci
[Abstract]


Cyclooxygenase-2 (COX-2) - A Therapeutic Target in Liver Cancer? Pp. 3305-3315
M. Breinig, P. Schirmacher and M.A. Kern
[Abstract]


MALDI Imaging of Biomolecules
Executive Editor: Michel Salzet


Editorial Pp. 3316


New Developments in MALDI Imaging for Pathology Proteomic Studies Pp. 3317-3324
M. Wisztorski, R. Lemaire, J. Stauber, S.A. Menguelet, D. Croix, O.J. Mathé, R. Day, M. Salzet and I. Fournier
[Abstract]


Mass Spectrometric Imaging of the Nervous System Pp. 3325-3334
S.S. Rubakhin, N.G. Hatcher, E.B. Monroe, M.L. Heien and J.V. Sweedler
[Abstract]


Recent Advances in Biological Tissue Imaging with Time-of-Flight Secondary Ion Mass Spectrometry: Polyatomic Ion Sources, Sample Preparation, and Applications Pp. 3335-3343
A. Brunelle and O. Laprévote
[Abstract]


A Snapshot of Tissue Glycerolipids Pp. 3344-3356
A.S. Woods, H.-Y.J. Wang and S.N. Jackson
[Abstract]




Abstracts


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Editorial: Resent and Future Therapies for the Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is becoming a major health problem worldwide, both because of the constantly rising number of patients and of the poor prognosis of this disease. Therapies adopted so far mainly fight the cancer by removing the tumor or the whole liver whenever possible by transplantation, or by destroying it either by alcoholization, radiofrequency ablation (RFA) or transarterial chemoembolization (TACE). The clinical outcome is often unsatisfactory and cancer recurrence or metastatic spread are common events that severely affect prognosis and survival.

No therapies are yet available to cure HCC, and this is rather surprising since for all other cancer forms, newly introduced pharmacological therapies have greatly improved survival. The concomitant liver cirrhosis where HCC commonly develops hampers the use of chemotherapeutics, which pose an increased risk due to the already reduced liver functionality.

As in other cancers, the idea is emerging that biological therapies may offer new hope for patients with HCC. In vitro results support this view and a better definition of the proper target of these drugs is gaining increasing importance, in order to improve their efficacy.

This issue deals with the important issue of therapies for HCC, including all the novelties introduced in the traditional but still important therapies, although particular attention will be devoted to the new strategies for biological therapies.

The first part of the issue discusses the therapies currently in use. However, this is not just an update on the current state of the art, although this would already be a relevant issue, but rather focuses on the future of these approaches, that will surely be milestones in the up-coming years, while new biological therapies acting as sharper bullets are being developed. In this regard, Lee Gordon and co-authors from the University of Vanderbilt, USA, have discussed the role of liver transplantation as a possible therapy for concomitant treatment of the HCC and underlying liver cirrhosis [1]. Michel Beaugrand and co-authors from the Hôpital Jean Verdier, France, have focused mainly on the use of locoregional therapies, that are currently widely used, and discuss the future applications of these techniques thanks to improved technologies [2]. The second part of the issue is dedicated to defining the biological and molecular bases regulating the biological properties of HCC, essential for the future development of target-oriented therapies. In this regard, Stefania Tommasi and co-authors from the National Cancer Institute of Bari, Italy, have discussed the role of genes involved in proliferation, metastatization and apoptosis as potential targets for future therapies [3]. Salvatore Antonaci and co-authors from the University of Bari, Italy, have reviewed the emerging role of the tissue microenvironment as a biological regulator of HCC behavior, in order to explain the role of different components potentially targeted by currently available biological therapies [4]. However, the next step will be to address how to develop a therapeutic strategy integrating the advances in more basic scientific fields in biomedical research. This is discussed in depth by Dipankers Chattopadyhay and co-authors from Newcastle University, UK [5]. There is no question that translational research is becoming highly relevant for the medical sciences, and this paper and the whole issue aim to set an example in this sense.

Finally, Gianluigi Giannelli and co-authors from the University of Bari, Italy, have reviewed the literature on the use of a promising class of drugs, tyrosine-kinase inhibitors targeted against the tissue microenvironment, as therapy for HCC [6]. Marco Breinig and co-authors from the University of Heidelberg, Germany, have discussed another interesting and intriguing class of molecules, COX-2 inhibitors, with an anti-tumor ability. This is an old class of molecules that has recently been reconsidered for use as therapies directed against the microenvironment components, that could provide new insight in the field of anti-cancer therapies [7].

References
[1] Koller FL, Geevarghese SK, Gorden LG. Liver Transplantation for Hepatocellular Carcinoma: Current Role and Future Opportunities. Curr Pharm Des 2007; 13(32): 3265-3273.

[2] Beaugrand M, Seror O. Transcutaneous treatment of hepatocellular carcinoma in patients with chirrosis present status and future developments. Curr Pharm Des 2007; 13(32): 3274-3278.

[3] Tommasi S, Pinto R, Pilato B, Paradiso A. Molecolar pathways and related target therapies. Curr Pharm Des 2007; 13(32): 3279-3287.

[4] Antonaci S, Giannelli G. Rationale for new drugs targeting the tissue microenvironment in patients with HCC. Curr Pharm Des 2007; 13(32): 3288-3291.

[5] Chattopadyhay D, Manas DM, Reeves HL. The Development of Targeted Therapies for Hepatocellular Cancer. Curr Pharm Des 2007; 13(32): 3292-3300.

[6] Giannelli G, Napoli N, Antonaci S. Tyrosine kinase inhibitors: a potential approach to the treatment of hepatocellular carcinoma. Curr Pharm Des 2007; 13(32): 3301-3304.

[7] Breinig M, Schirmacher P, Kern MA. Cyclooxygenase-2 (COX-2) - A Therapeutic Target in Liver Cancer? Curr Pharm Des 2007; 13(32): 3305-3315.


G. Giannelli
Dipartimento di Clinica Medica,
Immunologia e Malattie Infettive,
Sezione di Medicina Interna,
Policlinico, Piazza G. Cesare 11,
70124 Bari,
Italy;
E-mail: g.giannelli@intmed.uniba.it


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Liver Transplantation for Hepatocellular Carcinoma: Current Role and Future Opportunities
F.L. Koller, S.K. Geevarghese and D.L. Gorden

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. It represents the fifth most common cancer worldwide, and one whose incidence is on the rise. Liver cancer is the third most common cause of cancer mortality globally and thus a major health concern worldwide. Therapeutic options for this tumor include surgical resection, local ablative therapies, and systemic treatment. Liver transplantation has emerged as a highly effective treatment for patients with HCC, particularly in the setting of significant underlying liver disease. Current protocols in transplantation for this tumor utilize strict size criteria and staging (TNM classification) to select patients for this therapy. Selection criteria for liver transplantation for HCC that are accepted in the U.S. include: 1 tumor < 5cm, no greater than three tumor nodules, each less than 3cm in diameter 3) no macroscopic invasion of blood vessels or lymph nodes, and no extra-hepatic spread of tumor. Eligibility criteria and immunosuppression strategies are continuing to evolve in this field. Nonetheless, in appropriately selected patients, liver transplantation may provide a cure for HCC with survival rates equal to that of liver transplantation for end-stage liver disease (ESLD) from other causes. Liver transplantation has been established as one of the principal treatment modalities for this difficult disease.


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Transcutaneous Treatments of Hepatocellular Carcinoma in Patients with Cirrhosis: Present Status and Future Developments
M. Beaugrand and O. Seror

Hepatocellular carcinoma is in 90% of cases associated with cirrhosis and to preserve liver function while destroying the tumor is a main issue in these patients justifying the development of local percutaneous ablative therapies.

Alcoholization and radiofrequency ablation are the most widely used techniques of percutaneous ablation. Both have in common limitations linked to the size of the tumor and its situation and contra indications such has advanced liver disease and unpaired hemostasis.

Radiofrequency ablation despite specific contra indications such as a close vicinity of the colon has shown better results in term of tumor necrosis local recurrence and even survival.

For tumors less than 3cm in diameter results are comparable to those of surgical resection with a lesser morbidity. Due to this equivalence and the multiple contra indications of surgical resection in patients with cirrhosis radiofrequency ablation is becoming an increasingly popular technique for treating small hepatocellular carcinoma detected by screening.


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Molecular Pathways and Related Target Therapies
S. Tommasi, R. Pinto, B. Pilato and A. Paradiso

Hepatocellular carcinoma (HCC) is a frequent neoplasia which still misses a therapeutical gold standard. Recently, new acquisitions in cancerogenesis process evidenced the genetic and epigenetic alterations of genes involved in the different metabolic pathways of liver cancer suggesting that antibodies, small molecules, demethylating agents, etc. specifically acting against molecular target can be utilized alone or in combination in clinical practice. The main altered targets are: cell membrane receptors, in particular tyrosine kinase receptors, factors involved in cell signalling, specifically Wnt/β-catenin, Ras/Raf/MEK/ERK and PI3K/Akt/mTOR pathways, proteins linked to cell cycle regulation pathway (i.e. p53, p16/INK4, cyclin/cdk complex) or in invasiveness (EMT, TGFbeta) and proteins involved in DNA metabolism. Genetic or epigenetic changes in these molecules have been used in preclinical settings and, some of them also in clinical trials of phase II and III. This scenario opens new avenues for the prevention and the treatment of HCC. In the present review the main metabolic pathways and molecular alterations have been described together with recent advances in molecular and gene therapy.


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Rationale for New Drugs Targeting the Tissue Microenvironment in Patients with HCC
S. Antonaci and G. Giannelli

A better knowledge of the mechanisms underlying hepatocellular carcinoma (HCC) growth and spread is essential to improve the available treatment options. So far, the only therapies available for HCC are mainly based on tumor-destructive approaches, whereas no therapies are available to consolidate these invasive therapies or to cure the tumor. The fact that HCC develops on cirrhotic liver strongly limits the use of common anti-cancer drugs, but the need to find new therapies is strongly felt by clinicians. A large body of evidence suggests that the tissue microenvironment represents a potential target for therapies. Consistently, biological therapies such as inhibitors of the epithelial growth factor receptor (EGFR), are currently under investigation. Unfortunately, there is a discrepancy between the very promising experimental data and the results obtained in patients, although limited sample sizes and advanced stage of the disease could be important factors hampering a reliable judgment of the efficacy of such drugs. Nevertheless, a better identification of the molecular pathways involved in drug effectiveness as well as in HCC tumor progression indicates that the tissue microenvironment likely harbors the solution to the problem. In this review the role and the rationale of using biological drugs to target the microenvironment is discussed, taking into consideration new experimental advances in the field.


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The Development of Targeted Therapies for Hepatocellular Cancer
D. Chattopadyhay, D.M. Manas and H.L. Reeves

Present treatment options for hepatocellular cancer (HCC) are limited to those individuals with good liver function and early stage disease. Unfortunately this includes only a minority of patients, few of which are actually cured of their cancer. Over the last 15-20 years biotechnology has made a very significant impact on medical research, to the extent that we know very much more about the regulation of normal cell growth and death, as well as the mechanisms underlying its disruption in disease processes. This knowledge has and is being rapidly exploited by academic and pharmaceutical organisations, often in collaboration. The result is the development, testing and steady introduction of therapies that target specific abnormalities in cancer cells. Although the safety and effectiveness of the majority of these agents has yet to be established in cirrhotic patients with HCC, we are hopeful that we will shortly see an increase in effective treatment options available for clinical use this disease. This review focuses on aberrant cancer proteins and pathways relevant to HCC, as well as the novel therapies or strategies targeting them, that are currently in the development or testing stages.


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Tyrosine Kinase Inhibitors: A Potential Approach to the Treatment of Hepatocellular Carcinoma
G. Giannelli, N. Napoli and S. Antonaci

The increasing number of patients with hepatocellular carcinoma (HCC) and the highly unfavourable prognosis of the disease are two important reasons why more effort needs to be devoted to investigating other therapies able to block or reduce tumor progression and cancer metastasis. The underlying cirrhosis on which HCC develops limits the use of common chemotherapies, mainly because of their toxicity. Recently, great attention has been paid to a family of molecules that inhibits the tyrosine kinase (TK) receptors, because of their relevant role in activating intracellular pathways responsible for several biological activities of the HCC cells. In particular, proliferation, invasion, survival, apoptosis, are regulated by Erk1/2 and Akt pathways, that can be considered for this reason as potential therapeutic targets. Therefore, the idea is to fight HCC by blocking the molecular mechanisms exploited by the cancer to develop and to metastasize. The epithelial growth factor and the vascular endothelial growth factor receptors (EGFR and VEGFR, respectively) have been identified as the major targets for these new therapies. In this review the biological role of both growth factors in HCC will be discussed, together with the use of anti-EGFR and anti-VEGFR. The preliminary results obtained in vitro or in “in vivo” experimental models have been very promising, whereas the few studies conducted in patients have been not comparably satisfactory. This could be because of the limited number of patients and of their advanced stage of HCC, nevertheless the possibilities of using this family of drugs should be further explored, together with aspects contributing to a better understanding of the molecular mechanisms driving HCC progression.


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Cyclooxygenase-2 (COX-2) - A Therapeutic Target in Liver Cancer?
M. Breinig, P. Schirmacher and M.A. Kern

Targeting COX-2, a key-enzyme of the prostaglandin metabolism, for the treatment of cancer has been in the focus of researchers for about a decade. However, only recently has this topic been related to hepatocellular carcinoma (HCC). HCC is one of the most common cancers and a growing health problem worldwide. At present, only few promising treatment options are available, accentuating the urgent need for novel therapeutic approaches. Since the first report of COX-2 overexpression in HCC, several findings support the notion that selective COX-2 inhibition proves to be beneficial in this malignancy.

This review focuses on recent discoveries regarding the pro-tumorigenic potential of COX-2 in HCC and the functional effects of COX-2 inhibition on molecular mechanisms of this malignancy. Of clinical interest, promising data from in vivo experiments and case studies suggest a beneficial effect of COX-2 inhibitors for HCC- therapy. Detailed analysis of COX-2- activated pathways and related mechanisms may enable the evaluation and design of even more specific and combinatorial treatment approaches in the future.


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Editorial: MALDI Imaging of Biomolecules

Recent studies performed these last years on the proteome have demonstrated the usefulness of mass spectrometry The combination of mass spectrometry with classical biochemical methods has lead to the determination and identification of different proteins using proteomics studies in close relation with genome databases. Although such methods generally lead to the identification of novel proteins, they do not allow determining the cellular localization or regulation of peptide/proteins expression in tissues, cellular groups or single cells. New emerging technologies enable the development of alternative methodologies to address such questions so called MALDI imaging or SIMS.

This is the aim of this issue of Current Pharmaceutical Design devoted of 4 reviews on Imaging by mass spectrometry and their application in neuropathologies and clinical investigations.

This first article from Fournier team [1] is to highlight some of the more recent technological advances that have improved the efficiency of imaging mass spectrometry for clinical applications. Advances in the way MALDI mass spectrometry is integrated with histology, improved methods for automation, and better tools for data analysis are outlined in this review.

The second review from Sweedler team [2] is focused on MALDI imaging techniques available to investigate the nervous system, with particular focus on the capability of MSI to examine both normal and diseased brain function. An important investigative tool, MSI offers tremendous potential in fundamental studies of brain chemistry, localization of pharmaceutical compounds, and the discovery of biomarkers for different neuropathologies.

The third paper from Brunelle team [3] reviews the most recent advances in this field. After a short reminder of the basic physics involved, the instruments are described, as well as the primary ion sources, including the different cluster ion sources. The sample preparation methods are also described and compared, such as the matrix coating and the metal coating. The capabilities of the technique are finally illustrated with the most recent applications published in the last years.

The fourth paper from Woods team [4] is devoted to MALDI MS analyses in positive ion mode to examine and contrast positively charged phospholipids which are mainly located in the outer cellular membranes and compare their make up in the following organs brain, liver, kidney and heart.Phosphatidylcholines in particular are zwitterionic as they have both a phosphate and aquaternary amine, which allows them to interact with aromatic compounds as well as compoundscontaining carboxyl, guanidinium, phosphate and sulfate groups. Phospholipids propensity forinteraction explains why so many small molecules and therapeutic compounds are stored inadipose tissue, or are detected interacting with cellular membranes.

References
[1] Wisztorski M, Lemaire R, Stauber J, Menguelet SA, Croix D, Mathé OJ, Day R, Salzet M, Fournier I. New Developments in MALDI Imaging for Pathology Proteomic Studies. Curr Pharm Des 2007; 13(32): 3317-3324.

[2] Rubakhin SS, Hatcher NG, Monroe EB, Heien ML, Sweedler JV. Mass Spectrometric Imaging of the Nervous System. Curr Pharm Des 2007; 13(32): 3325-3334.

[3] Brunelle A, Laprévote O. Recent Advances in Biological Tissue Imaging with Time-of-Flight Secondary Ion Mass Spectrometry: Polyatomic Ion Sources, Sample Preparation, and Applications. Curr Pharm Des 2007; 13(32): 3335-3343.

[4] Woods AS, Wang H-YJ, Jackson SN. A Snapshot of Tissue Glycerolipids. Curr Pharm Des 2007; 13(32): 3344-3356.


Prof. Michel Salzet
Membre de l’Institut Universitaire de France,
FRE CNRS 2933,
Université des Sciences et Technologies de Lille.
59650 Villeneuve d’Ascq
France
E-mail: michel.salzet@univ-lille1.fr


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New Developments in MALDI Imaging for Pathology Proteomic Studies
M. Wisztorski, R. Lemaire, J. Stauber, S.A. Menguelet, D. Croix, O.J. Mathé, R. Day, M. Salzet and I. Fournier

With new emerging mass spectrometry technologies, it can now be demonstrated that direct tissue analysis is feasible using matrix-assisted laser desorption/ionization (MALDI) sources. A major advantage of direct MALDI analysis is to avoid time-consuming extraction, purification or separation steps, which have the potential for producing artifacts. Direct MALDI analysis of tissue sections enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. With automation and the ability to reconstruct complex spectral data using imaging software, it is now possible to produce multiplex imaging maps of selected biomolecules within tissue sections. Thus, direct MALDI spectral data obtained from tissue sections can be converted into imaging maps, a method now known as MALDI-imaging. MALDI-imaging combines the power of mass spectrometry, namely exquisite sensitivity and unequivocal structural information, within an intact and unaltered morphological context. Critical improvements to increase image resolution are presented in this manuscript e.g., solvent treatment, new solid ionic matrices, gold sputtering, nickel support or laser focalization. One of the most important developments is the ability to carry out either direct MALDI analysis or MALDI imaging on paraffin tissue sections, thus opening the path to an archival “gold-mine” of existing pathology samples to proteomic analysis. These developments provide new avenues for biomarker hunting and diagnostic follow-up in the clinical setting. Further developments in MALDI-imaging of specific targets provide an added dimension, as validated disease-marker-gene RNA transcripts can be analyzed along with their translation by targeting their specific protein products or metabolites. Disease/health states will thus be closely molecularly monitored at protein and nucleic acids levels, with a single technique. Taken together, MALDI imaging will become a key tool for pathology proteomic studies.


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Mass Spectrometric Imaging of the Nervous System
S.S. Rubakhin, N.G. Hatcher, E.B. Monroe, M.L. Heien and J.V. Sweedler

Mass spectrometric imaging (MSI) integrates multiple fields of analytical and biomedical research with the goal of generating chemical maps that present the identity and location of the elements, molecules, and molecular complexes that comprise biological structures. Rapid advances in the development of MSI, which include a broad range of sampling and mass spectrometry strategies, allow the increasingly information-rich creation of chemical images of structurally complex tissues, individual cells, and even single chromosomes. Here we describe a variety of MSI techniques available to investigate the nervous system, with particular focus on the capability of MSI to examine both normal and diseased brain function. An important investigative tool, MSI offers tremendous potential in fundamental studies of brain chemistry, localization of pharmaceutical compounds, and the discovery of biomarkers for different neuropathologies.


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Recent Advances in Biological Tissue Imaging with Time-of-Flight Secondary Ion Mass Spectrometry: Polyatomic Ion Sources, Sample Preparation, and Applications
A. Brunelle and O. Laprévote

Recent technological and methodological improvements have greatly enhanced the sensitivity of the Time-of-flight Secondary Ion Mass Spectrometry (TOF-SIMS), thus making this technique now very attractive in the field of molecular imaging of biological samples such as tissue sections or cells. This paper reviews the most recent advances in this field. After a short reminder of the basic physics involved, the instruments are described, as well as the primary ion sources, including the different cluster ion sources. The sample preparation methods are also described and compared, such as the matrix coating and the metal coating. The capabilities of the technique are finally illustrated with the most recent applications published in the last years.

http://www.bentham.org/cpd/contabs/brunelle/images


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A Snapshot of Tissue Glycerolipids
A.S. Woods, H.-Y.J. Wang and S.N. Jackson

The lipid membrane is the portal to the cell and its first line of defense against the outside world. Its plasticity, diversity and powers of accommodation in a myriad of environments, mirrored by the varied make up of the cells it protects, are unparalleled. Glycerophospholipids are one of its major components. In cell membranes the extracellular layer is mainly made up of positively charged gly-colipids, while the intracellular one’s main components are negatively charged. Advances in mass spectrometry have allowed the direct probing of tissues, and thus a direct approach to probing membranes make up was developed. Until recently most studies have focused on proteins. An overview of the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) for the direct analysis of phospholipids in various tissue is presented. Molecular ions corresponding to phosphatidylcholines, sphingomyelin, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols and sulfatides were mapped.