Cancer is innumerable group of disease with as many different manifestations as there are tissues and cell types in the human body, involving numerous endogenous or exogenous carcinogenic agent and various etiological mechanisms. One most common thing from all of these disease are certain biological properties of the cells that compose the tumours, including unregulated cell growth, impaired cellular differentiation, invasiveness and metastatic potential. We can see that there are significant improvements in the diagnosis of and advance in the therapy of specific cancers. However, cancer continues to represent a major medical concern, delimiting longevity and the quality of human life. This is the fact that current estimates indicate that from every 3 people one person is victim of cancer during their lifetime.
Hundreds of mitochondria are present in eukaryotic cells, each of them have several copies of mitochondrial genome. When homoplasmic mutation(somatic acquired mutation) occurs in the mitochondrial DNA it expand to wide range of mitochondrial genome, within the same cell and it have been identified in wide variety of cancer. Some of these mutations are contributed to cancer cell phenotypes by number of mechanism such as an acceleration of mitochondrial dysfunction, increase generation of free radical, conversion of oxidative into glycolytic metabolism, and dysregulation of apoptosis.
Novel Molecular target for cancer Drug discovery
Within the last three decades, molecular oncology has revealed that the genetic and epigenetic alteration had led to the multistage process of cancer growth and progression.
By understanding the molecular mechanism, the more efficacious and less toxic drug approach have been designed to inhibit the specific proteins or abnormal pathway which are generally expressed in cancer cell. This approach, designated as "Target therapy" is applied to many scientific research to discover new targets and to develop new anticancer drug candidates. (Collins and Workman 2006)
The fundamental steps involved in drug discovery are drug identification and validation, clinical identification and clinical studies of Phase I-III, hit-to-lead and lead optimisation.
Since 1955 compounds of both synthetic and natural origin were screened in a panel of cancer cell lines and of mice tumours such as the L1210 leukaemia model.
In 1976 the cancer xenografts were successfully introduced in nude mice for screening purposes and their observation has generated an approach for cornerstones of cancer therapy, including taxol and antracyclines, with the molecular
mechanism of action of these drugs (actually, the target) discovered several years after the identification of the active comstituents. As an example, by understanding the molecular mechanism of DNA and DNA-interacting proteins give clear concept of mechanism of action of drugs targeting topoisomerases and DNA itself (e.g. etoposide and platinum derivatives) (Suggitt, et al. 2005).
It has been shown in the last 25 years of molecular oncology that the precise combination of genetic alteration is responsible for cancer arise in a susceptible cell.
The key regulators of vital cell functions, especially cell cycle and proliferation, apoptosis and cell motility are involve in genetic alteration.
For cancer drug discovery, proteins abnormally expressed in cancer cells as a consequence of genetic alterations may represent potential targets. The theory behind target driven drug discovery have been promoted by the elucidation of the roles of many kinases, including receptor kinases and signaling kinases, along with the proof that these enzymes are specifically "druggable" targets.
The following linear sequence of steps are described as the "targetcentric" approach to the drug discovery, starting from the identification of a protein altered in cancer cells, followed by the development of an assay assessing the anticancer drug discovery and development biological activity of the target, screening of compounds inhibiting the target and, after reiterated cycles of optimization and re-testing, identification and selection of inhibitors, with adequate properties (in terms of potency, specificity, drug-like properties, preclinical tolerability) to be tested in animals and in humans for antitumor efficacy and possible toxicological liabilities.(Sager, et al. 2003)
The most fundamental problems occurring during the cancer drug discovery are toxicity and selectivity because the drugs in use are highly cytotoxic and not have selectivity between tumour cells and normal cells. Therefore, the novel cancer therapy requires specificity that will very toxic to cancer cells and little or non toxic to normal tissues. Recent innovation in cancer cell biology offers new hope for targeting only neoplastic cells by advances in recombinant DNA technology. In addition to molecular target, DNA recombinant technology has facilitates the investigation of new target for Drug design and development, includes the functional expression.
An
Currently one of the most effective drugs for the cancer therapy is cisplatin. It is highly mutagenic and carcinogenic in both in vitro and in vivo experimental models and causes primarily intrasrand DNA-DNA cross links.
A number of analogues of cisplatin are available and one of these, carboplatin (CDDCA) is also use clinically.
Around 298,000 new cancer incidences have been diagnosed each year in the most recent time period in both men in UK.. More than 200 types of cancer have been identified, but among them lung, prostate, breast and colorectal cancer account for over half of all new cases. The most common cancer in UK is the breast cancer.
Human Melanoma Development and Progression
The incidence of melanoma is increasing significantly in developed countries. The several factors should be considered which leads to melanoma those are racial and hereditary.
Ultraviolet (UV) Light
The uv rays present in the sunlight has been implecated as probable cause of melanoma developement. UV could cause DNA instability , inhibit antioxidants and suppress the immune system.[ Cifone MA et al.,(1981) Fuchs J et al., (1990) Penn I (1985)]. The expression of transforming growth
factor (TGF) and nerve growth factor on
melanocytes which is generally induced by UV light.[ Ellem KAO, et al., Peacocke M, et al.,(1988)]. Recently animal experiment has proved that UV either causes or contributes to melanoma development.
Suppressor Oncogenes and Genes
ras
Human melanoma involves expression of three ras genes (N-ras, Ha-ras, and Ki-ras). N-ras appears to be the most responsible for the primary melanomas.[ Platz A et al., (1994)]
p53
Both mutation and overexpression of this tumorsuppressor gene leads to melanoma.
p16
p16 is a product of the cyclin-dependent kinase (CDK)
N2 gene involved in the G1/S checkpoint of the cell cycle. When mutations and deletions of the p16 cause cutaneous malignant melanomas Methylation of the promoter region of the gene allows suppression mechanism of CDKN2A.In general disruptions of CDKN2A:CDK4 pathway play a major role in melanoma development.
Factors responsible for invasion and metastasis of Melanoma
First of all we have to investigate the mechanisms involved in metastasis is necessary to controlthis neoplasm. The changes of matrix metalloproteinase activity and characteristics of vasculature in malignant melanoma are the two key factors in local invasion and hematogeneous metastases of melanoma.
Role of Matrix metalloproteinase in melanoma progression
Degradation of basement membranes and extracellular matrix (ECM) is an fundamental step in cancer invasion and metastasis.
Matrix metalloproteinase (MMP) and their tissue inhibitors TIMP) facilitates this process. MMPs are zinc-dependent endopeptidases accountable for the degradation of extracellular metrix components. To date,about more than 15 human MMPs have been cloned and characterized. In relation to malignant melanoma, increased expression of MMP-1, MMP-2, and MMP-9 was shown to correlate with an invasive phenotype. In a xenogtaft model, metastatic capacity of melanoma cells is correlated with increased expression of MMP-2.
Significance of vasculature in prognosis of malignant
melanoma
In the early stages, malignant melanoma develops hematogeneous metastases. Therefore, investigation of vascularity and fragility of blood vessels in this neoplasm is necessary. Generally, a smooth muscle actin (SMA) is expressed in vascular smooth muscle cells, myoepithelial cells and some other elements. Taniguchi's group found that blood vessels with redused expression of SMA in malignant melanoma. They also found that melanoma cells released a factor which inhibits the expression of aSMA [69]. Recent study on melanoma research found reduced expression of calponin-h1 (CNh1) and caldesmon (CD) in the blood vessel walls in melanoma tissues. CNh1 and CD are actin- and calmodulin-binding proteins which are key componant in the structure and function of smooth muscle. The above mentioned data suggest that blood vessel walls in the lesions of melanoma are fragile, and this fragility may contribute to the development of hematogeneous metastasis.
Treatment of cancer
The aim of an ideal cancer treatment is the removal or destruction of all cancer cells. But in many cases this may not possible because the tumour has involved vital organs or has spread throughout the body. Cancer can be treated by many therapies such as radiation therapy, chemotherapy, surgery and other treatment methods includes angiogenesis inhibitors therapy, gene therapy, laser treatment, photodynamic and targeted therapy. Most of these therapies are used these days.
Anti antigenic drugs: One of the hallmarks of cancer is angiogenesis, the formation of new blood vessels from the existing vascular bed.
Radiation therapy is another widely used method for destroying tumour cells but there are often problems in applying a high dose to kill tumour cells without destroying the surrounding normal tissues and also associated with radiation risk. Because of these difficulties cytotoxic drugs and or methods for stimulating the body's own immune system are in use and have the added advantage of potentially being able to destroy tumour cells that have already spread away from the primary tumour. The replacement of defective tumour suppressor genes or insertion of a gene that is thought to novel approach for cancer therapy.
SAHA (suberoylanilide hydroxamic acid)
A number of histone deacetylase inhibitors have been identified during past decade that induced tumour cells in culture and in cancerous animal to undergo growth arrest, differentiation and apoptosis. Acetylation and deacetylation of histone plays a significant role in regulation of gene expression. Mainly two class of enzymes are involved in determining the degree of acetylation of histones, Histone acetyltrasferase (HAT) and Histone deacetylase (HDAC).
SAHA administered either intravenously or orally which found that accumulation of acetylated histones in peripheral mononuclear cells and tumour cells. In Phase I clinical trial, orally-administered SAHA(vorinostat) in patients with advanced cancer identified a maximum tolerated dose of once-daily 400 mg and twice-daily 200 mg for continuous daily dosing, and twice-daily 300 mg for three consecutive days per week dosing. Clinical anti-cancer activity was observed at 43% oral bioavailability and also shows linear pharmacokinetics. A subsequent Phase II study explored a range of doses and found that once-daily 400 mg vorinostat had the greatest safety profile and demonstrated activity in heavily pretreated patients with cutaneous T-cell lymphoma (CTCL).
Metal binding moiety
Carbon linker
Capping group
Recently Suberoylanilide hydroxamic acid (SAHA, Vorinostat, ZolinzaTM) has gained FDA approval for the treatment of advanced cutaneous T-cell lymphoma (CTCL). SAHA inhibits the activity of histone deacetylase enzyme in proteins, which are correlated to a variety of cancers. SAHA is the first HDAC inhibitor (HDACi) to get FDA approval, several other small molecules that inhibit HDAC proteins are currently in clinical trials for cancer therapy. Unique characteristics of HDAC inhibitors such as a metal binding moiety, a carbon linker, and a capping group . Based on crystallographic studies, the capping group is solvent-exposed and interacts with amino acids close to the entrance of the active site, while the metal binding moiety resides in the interior part of protein and complexes the metal ion involved in catalysis. The linker serves the capping position and metal binding groups suitable for high-affinity interactions with proteins. With a7 modular framework and application toward cancer treatment, HDAC inhibitors are possible targets for future drug design.
SAHA analogs with substituents adjacent to the capping group demonstrated potent nanomolar inhibitors. In contrast, SAHA analogs with substituents adjacent to the hydroxamic acid confirmed micromolar IC50 values. The combined data suggest that substituents are tolerated along the linker chain but potency declines when positioned near the metal binding moiety. Because simple isoform selectivity has been reported with HDAC inhibitors bearing substituents along the linker a systematic assessment of substituent tolerance along the linker chain will point future HDACi design. The effect of substituents at additional positions along the linker chain is currently under investigation.
Acetyl groups on the lysine tails of DNA associated histone proteins interact with the phosphate backbone of DNA and in doing so allow access of transcription factors to DNA. Deacetylation of histone proteins by HDACs results in compacted and therefore, unaccessible chromatin. In the presence of HDAC-inhibitors, chromatin remains in an opened configuration, allowing transcription factors to reach DNA promoters and facilitate transcription of tumor suppressor genes that check the growth of cancer cells. HDAC-inhibitors including SAHA have a wide variety of functions important for treating cancers including induction of apoptosis or differentiation, cell cycle arrest, and inhibition of angiogenesis.
Future guidelines
Gene micro array profiling has been helpful in identifying a number of target genes for determining both sensitivity and resistance to SAHA and other HDAC-inhibitors. Skin biopsies were used to study biomarkers predictive of SAHA sensitivity and resistance cancer cells including malignant Tcells become resistant to the HDAC-inhibitors and one of the most common reason is development of resistance. Therefore, single agent is not enough to effective against such type of resistance. Thus, rational combinations of SAHA with other agents are conventional approach to solve this problem.The invention of HDAC-inhibitor biology and clinical applications is just beginning and the future seems very bright for the DNA demethylating agent.
Accumulation of acetylated histones in normal peripheral mononuclear (PMN) cells is the biomarker of HDACi. This marker does not show a relationship with clinical efficacy but it gives an indication that accumulation of the HDACi at the site. For example, a single oral dose of SAHA, 400 mg (OD) will induce PMN cells to accumulate acetylated histones in 2-4 h which takings to pretherapy levels in 10-14 h.
B16 (murine melanoma cell line)cell culture
In vitro cytotoxicity assessment has recently been become popular as a primary screening method for evaluating the antitumor activities of various chemicals. For example, cisplatin and other platinum analogue have been shown to cause their cytotoxic effects on tumor cells in culture.
Recently, the cytotoxic effects of various chemicals and natural substances on malignant tumor cells in culture have been extensively studied as a primary screening for their antitumor activities, and hence it seems important and necessary to confirm the connection between the in vitro cytotoxic and in vivo antitumor activities. In this respect, it seems helpful for assessment of the antitumor activities of cisplatin and SAHA (suberoylanilide hydroxamic acid) to understand cytotoxic profiles. For this purpose, the in vitro cytotoxic effect of SAHA (suberoylanilide hydroxamic acid) on murine tumor cells was further investigated.
Many cell types have been shown to release inhibiting or stimulating substances into their culture medium [l-3]. These activities can regulate growth of cells in culture, but can also explain invasive capacities of cancer cells in vivo. We previously showed that mitogenic and cytotoxic soluble factors were released into serum-free media conditioned by different lines of mouse B16 melanoma cells in culture [3]. The cytotoxic activity was concentrated in ultrafiltrate Ul (Mw < 1000 Da) prepared from media conditioned by a heterogeneous parental cell line (B16) and two derived cloned cell lines, containing either non pigmented (NPB16) or pigmented (Pq16) cells. Ultrafiltrate Ul of the medium conditioned by non pigmented B16 cells (NPB16 CM IA) was the most cytotoxic [3]. Interestingly, this line was the less metastatic and less tumorigenic in the mouse [4]. Ideal experimental conditions needed for the production of the cytotoxic factors as well as some of their characteristics. The conditioned media were generally tested on non pigmented B16 cells, which were the most sensitive line. First, we have to investigate the effects of non pigmented B16 CM Ul on cell proliferation and differentiation as a function of dilution, duration of treatment or of medium conditioning. Then, several agents were added during the conditioned media (CM) preparation, in order to evaluate their effect on the rate of cytotoxicity of these NPB16 CM Ul.
TABLE I. Drugs That Inhibit Topoisomerases I and I1
Enzvme
Topoisomerase I1 Doxorubicin
Daunorubicin
4-Epidoxorubicin
Idarubicin
Mitoxantrone
MAMSA
Etoposide
Teniposide
Intercalating Agents
Epipodophyllotoxins
Topoisomerase I Topotecan Camptothecin
Topoisomerase 1 and I1 Dactinomycin Intercalating Agent
Angiogenesis inhibitors:
Reduce microvascular growth or block proangiogenic pathways
(Bevacizumab, ixabepilone)
Progrowth
Antiapoptosis
Proangiogenesis
Tumour cell
Microtubule stabilizing agents:
Halting cell-cycle by stabilizing microtubules (taxanes, epothilones)
Nucleus
Topoisomerase Inhibitors:
Promote cell-cycle arrest by
inducing DNA brackage.
(anthracyclines, capecitabine,
gemcitabine, platinum agents)
Growth-factor inhibitors:
Competitively inhibit
growth-factor binding and
promote ADCC (trastuzumab,
cetuximab, pertuzumab)
RTK inhibitors:
Block downstream
oncogenic pathways
(lapatinib, sunitinib
Endothelial cells on vasculature
Figure. Schematic diagram of the mechanisms of action of recently developed cytotoxic and biologic combination treatment regimens for several metastatic cancer. Term used X = point of inhibition; ADCC = antibody dependent cellular cytotoxicity; RTK = receptor tyrosine kinase.
NMR
Drug candidate and macromolecular targets are the indispensable tool in drug discovery and developement. NMR spectroscopy providing structural information on low molecular weight drug cancidates and on their ligand binding domain of receptor and enzyme. NMR spectroscopy offers a non-invasive insight into metabolic processes in cellular preparationsand isolated organs.
Animal models are used in a number of phases of the
drug development process. Transgenic animals are being more and more used for various reasons, e.g. the selection and validation of a potential drug target in early stage. In later phases drugs
are profiled using animal models of human disease to evaluate the mechanisms, the pharmacokinetic
and pharmacodynamic aspects, and the safety
protocol of a drug candidate.
In 1971, Damadian found new application of detection of tumours. He described that the T1 relaxation time in various types of cancer tissues was significantly longer than in the adjacent normal tissue.MR signal might provide classification criteria adequate to discriminate benign from malignant tissue masses and also gives detailed staging of the tumour. Recognition techniques will enhance the discriminative power when it work through combination of imaging and spectroscopy (i.e. MRI, MRS). In preclinical assessment, MRI can be useful to assess the tumour volume and also in physiological parameters such as neovascularisation and tumour metabolism.
MRI has been used by wide range of research groups to quantify the efficacy of drugs on tumour growth. The somatostatin analogues octreotide and bicalutamide showed significant reduction in the growth of Dunning prostate R3327 tumours in
the rat.
Abstract
Cancer is a diverse class of disease with as many different manifestations as there are tissues and cell types in the human body, involving myriad endogenous or exogenous carcinogenic agent and various etiological mechanisms. So there are numerous
