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Cancer is commonly referred to disease which normal cells undergo abnormal cell proliferation and differentiation. These abnormalities are due to mutations to cell genes, thus producing oncogenes and causing the tumor suppressor genes to lose its functions. Nowadays cancer has become a common disease as many people have been diagnosed with it and is considered a major cause for mortality worldwide. World Health Organization has reported that cancer has caused 7.4 million mortalities in 2004. In Peninsular Malaysia, almost 70 000 new cases were reported from 2005-2008 (MAKNA, 2006). Colorectal cancer is the third most common cancer worldwide and causing half a million of mortality annually, as reported by World Health Organization (WHO, 2009). Colorectal cancer has an almost identical pathophysiology as other types of cancer but the origin starts at the colorectal of the human body. In Malaysia, colorectal cancer is the second most common cancer reported according to the National Cancer Registry (NCR) and the cases cover 13.2% of overall cancer cases that have been reported (NCR, 2006).
Nowadays chemotherapy is synonymous with cancer, despite other cancer managements, such as surgery and immunotherapy. Chemotherapy aims at interfering with cancerous cells progression using chemical agents. There are numerous chemotherapy drugs available in the market for cancer treatment. The chemotherapy drugs can be used as single agent chemotherapy and combination chemotherapy, which means two or more chemotherapy drugs, are used together for management of cancer. However, chemotherapy drugs, either used as single or in combination, present various adverse effects such as bone marrow suppression as stated by American Cancer Society (ACS, 2010), nausea and vomiting (de Boer-Dennert et al., 1997).
Therefore, further drug discovery is conducted to discover new anticancer drugs that elicit less adverse effects, more selective and effective agents (Nam andÂ Parang, 2003). Researchers have turn to natural sources for this purpose. Nature is abundance have such a wide biodiversity (da Rocha et al., 2001). Plants with such diversity have helped in drug discovery. For example, a well-known chemotherapy drug paclitaxel is isolated from bark of yew tree Taxus brevifolia and its derivatives docetaxel, has been isolated from the leaves of Taxus baccata (da Rocha et al., 2001).
Other than plants, microorganisms also play a major role in anticancer drug discovery from natural products. Endophytes, which are microorganisms found in plants and can be described to have symbiosis interaction with its host (Tadych and White, 2009).
The objectives of this study are:
To grow and extract selected endophytic fungi (PA1US1, PA1ML2 and PA1ML3) which have been isolated from Ixora congesta.
To determine the anticancer activity of the endophytic extracts against colon cancer cell lines.
To determine the selectivity of endophytic extracts against colon cancer cell lines as compared to a normal cell line.
The human body is made up of numerous cells. These cells proliferate, differentiate and die in an orientated manner. The human cells undergo normal growth and maturation during the early years of life, and during tissue repair. However, there may be some abnormalities during the process, resulting in uncontrolled proliferation of cells. Therefore, cancer is characterized by a change or deviation in the control mechanisms of cell survival, proliferation and differentiation (Chu and Sartorelli, 2007). Cancer cells are said to be normal cells having damaged genes that directly regulates their cell cycles. Genetic alterations or mutations of DNA of the normal cells produce oncogenes, and cause the loss of function of tumor suppressing genes (Hanahan and Weinberg, 2000).
Cancer has been a major cause of death worldwide as it accounted for 7.4 million deaths, which is 13% of all deaths in 2004, as reported by World Health Organization. This has been quite a concern as the death from cancer is projected to increase by estimation of 12 million deaths in year 2030 (WHO, 2009). In Malaysia, nearly 70 000 cases were diagnosed between 2003 and 2005 (MAKNA, 2006) and a total of 21 773 cases were diagnosed among Malaysians in Peninsular Malaysia in 2006 (NCR, 2006). These data show that cancer will remain and escalate as the lead cause of mortality not just in Malaysia, but worldwide.
Colon cancer has been the third most common cancer following lung cancer and breast cancer, with more than 940 000 cases reported annually worldwide causing nearly 500 000 mortality ever year (WHO, 2003). Even in the U.S., colon cancer is the third most common cancer among both men and women (ACS, 2009). Up to year 2006, about 70 270 men and 68 857 women were diagnosed with colon cancer and the mortality statistics were 26 801 in men and 26 395 in women, in the U.S. (CDC, 2010). In Malaysia, according to the National Cancer Registry, colon cancer has become the second most common cancer with up to 13.2% of overall cancer cases (NCR, 2006). From these cases, Age Standardized Incidence Rate (ASR) for cases involving Chinese were the highest, 21.4 per 100 000 population, and were lower in Malays and Indians with ASR of 11.3 per 100 000 and 9.5 per 100 000 respectively (NCR, 2006).
2.1.1 Colon Cancer
Colon cancer, as the name suggest, is a cancer that starts in the area of the colon. It is suggested that the cause of colon cancer is mutation of the adenomatous polyposis coli (APC) gene. Patients that inherit a defective APC allele are diagnosed of having familial adenomatous polyposis (FAP) syndrome which may cause the development of many polyps, or adenomas beginning after puberty (Cappell, 2005). Polyps can be defined as the benign outgrowth of the lining of the colon and rectum (NCSM, 2010). These polyps may lead to the formation of adenocarcinomas, inevitably resulting in colon cancer. Another pathogenesis of familial colon cancer syndrome is hereditary nonpolyposis colon cancer (HNPCC) (Cappell, 2005). This particular syndrome is caused by mutations of one of the mismatch repair genes, such as hMLH1 and hMSH2 genes. Mismatch repair genes are genes that encode for mismatch repair enzymes that recognize and correct errors in nucleotide matching of complementary chromosome strands (Cappell, 2005). The accumulation of the mutations further leads to mutation of the tumor suppressor genes and oncogenes, such as p53 genes which initiates the formation of colon cancer (Cappell, 2005).
There are 5 stages involved in colon cancer progression. The stages and descriptions are shown in Table 2.1.1 and Figure 2.1.1
Table 2.1.1: Stages of colon cancer.
The cancer is very early. It is found only in the innermost lining of the colon or rectum.
The cancer involves more of the inner wall of the colon or rectum.
The cancer has spread outside the colon or rectum to nearby tissues but not to the lymph nodes. (Lymph nodes are small, bean-shaped structures that are part of the body's immune system.)
The cancer has spread to nearby lymph nodes but not to other parts of the body.
The cancer has spread to other parts of the body. Colorectal cancer tends to spread to the liver and/or lungs.
Figure 2.1.1: Sites according to the stages of colon cancer.
(Adapted from Gani, 2009)
There are various ways for the management of cancer. Chemotherapy is one of them and is commonly applied. Chemotherapy basically refers to the use of chemical agents to intervene the progression of cancer. It is usually inserted into the body, through oral route in the form of pills, or intravenously by injections. Therefore, chemotherapy is said to exert systemic effect to the body of patients. The aim of chemotherapy is to kill cancer cells by interfering with the process of cell division because cancer cells undergo active cell division as compared to most normal cells. The targets of the chemical agents are either the proteins involved during cell division, or directly damaging the DNA of the cells (Cancer Research UK, 2010). Chemotherapy can be given as a single agent or in combination chemotherapy. Single agent chemotherapy means using a single chemical agent from any classes of chemotherapy drugs for the management of cancer, while combination chemotherapy refers to the use of several chemical agents, consisting of several classes of chemotherapy drugs in regimen. For example, it is found that combination of Bevacizumab with Oxaliplatin, Fluorouracil, and Leucovorin (FOLFOX4) improves survival extent of patients previously treated for metastatic colorectal cancer (Giantonio et al., 2007).
Chemotherapy is associated with numerous side effects. These side effects occur because chemotherapy aims and kills actively dividing cancer cells, and exerts systemic effect. Some normal body cells also divide actively which is essential for the body to maintain its function. The normal cells that are most likely to be damaged are bone marrow, cells of hair follicles, cells lining the digestive tract, and cells lining the reproductive tract (ACS, 2010). Patients that undergo chemotherapy may develop bone marrow suppression, hair loss, appetite and weight loss, constipation, diarrhea, liver and kidney damage, or even worse, permanent organ damage and secondary cancer as long term side effects. However, nausea and vomiting was found to be the two most distressing adverse effects of chemotherapy with the use of serotonin antagonist (de Boer-Dennert et al., 1997).
Resistance of cancer cells towards chemotherapy has long time being researched intensively. Cancer cell resistance is not the result of a single gene alteration. Cancer cells also contain multiple genetic defects such as mutations, translocations and other types of defects. Epigenetic aberrations, such as alteration of DNA methylation may also occur. Synergism by these defects may result in cancer cell resistance as the cells try to maintain homeostasis and to cope with the effects of chemotherapy or radiotherapy (Perez-Plasencia and Duenas-Gonzalez, 1996). Cancer stem cell have the ability to self-renew indefinitely, while maintaining their ability to generate both tumorigenic (TG) and non-tumorigenic (NTG) cells and it was discovered that colon cancer cells developed after the exposure of chemotherapy agent, cyclophosphamide (Dylla et al., 2008).
2.3 Drug Discovery from Natural Products
Research of new drugs is a never ending process. Although current drugs are providing therapeutic benefits, further research must be conducted to find alternative drugs that are more specific, have less adverse effects. This also includes improving ways to produce drugs in a more economical way with higher yield.
Drug discovery and development have evolved a long way and incorporated new technological advancements. Over centuries, natural products have been the major source of new drugs. However, with the advancement of technology, proteomics and genomics discoveries, high throughput screening, combinatorial chemistry and other technologies have been implemented in current drug findings (Sofia, 2004).
Focusing on anticancer agents, the aims of drug discovery are to find less toxic, more selective, and more effective agents (Nam and Parang, 2003). The findings of new anticancer agents resolve around some aspects, which are either discovering new compounds from natural sources or modifying structure of known anticancer agents (Ma and Wang, 2009). In the past, synthetic products such as alkylating agents were the only chemotherapy agents of choice. However, synthetic compounds exhibit tremendous side effects as these agents are not cancer cell specific, thus injuring rapidly dividing normal cells. Scientists have begun the search for selective anticancer agents that have less side effects when compared to the synthetic compounds.
Even with all the technologies and synthetic compounds produced, natural products have been a major source for drug discovery and development for over centuries. Analysis and understanding of natural products have become a breakthrough in the development of purified and defined compounds referred to as dose-controlled medicines (Rishton, 2008). It is analyzed that 60% of approved drugs are derived from natural compounds (da Rocha et al., 2001). Nature is a great source of finding new therapeutic candidate compounds because nature is abundance and has great chemical diversity (da Rocha et al., 2001). There are three broad categories of sources of natural products which are plants, marines and microorganisms.
2.3.1 Natural Products in Plants
Plants have long been exploited as a source for drug discovery. Many people have used them as traditional medicines and for other purposes such as dyes and food additives for centuries. Even until today, medicinal herbs are still being studied and used for treating various illnesses. It has been estimated by the World Health Organization that approximately 80% of the worldâ€™s inhabitants rely mainly on traditional medicines for their primary health care (Newman et al, 2000).
Now, as biotechnology evolved and entered the picture in drug discovery, especially associated with plants, more advantages are offered in terms of production scale and economy, product safety, ease of storage and distribution (Ma et al, 2005). This gives opportunity for researchers and pharmaceutical companies to supply low-cost drugs and vaccines.
However, deriving an agent from nature resources do not come without difficulties. For instance, consider during the introduction of drug paclitaxel, which is originally isolated from bark of yew tree Taxus brevifolia. Researchers and pharmaceutical companies were in conflict concerning the environment because numerous trees and forests will be chopped down if they were to meet the drug demand. Eventually, after some years, a semi-synthetic derivative of paclitaxel, docetaxel, has been isolated from a renewable source, which is the leaves of Taxus baccata.
Various natural products from plants have been marketed around the world. Examples of the products areÂ Cynarin which is a plant chemical found in the common artichoke (Cynara scolymus) for liver problems and hypertension; quinine, which was discovered in a rainforest tree (Cinchona ledgeriana) as antimalaria; and digoxin found in Digitalis lanata as cardiotonic (Taylor, 2000). Table 2.3.1 shows some anticancer agents that are undergoing Phase III/IV trials.
Table 2.3.1: Some anticancer agents of status Phase III/IV derived from natural resources and their cancer treatment.
Leukemia, lymphoma, breast, lung, paediatric solid cancers and others
Breast, lymphoma, germ-cell and renal cancer
Ovary, breast, lung, bladder, and head and neck cancer
Colorectal and lung cancer
(Adapted from da Rocha et al., 2001)
2.3.2 Natural Products in Marines
Sea covers about 70% of the earth, making it a valuable resource for drug discovery. Until now, marine source is underexploited. Most drug discovery programs use samples only near the surface regions. However, there are more diverse and abundant microorganisms and makrofauna deep within the oceans. Such organisms that have associated with post- and current drug discovery are marine microbes, algae and invertebrates. More than 15 000 structurally diverse natural products with various and different bioactivities have been discovered since 1970 (Baker et al, 2007).
However, there are certain drawbacks in using marine as a source for new drug findings. As stated above, the inability to search deep into the ocean for further diversity is a major drawback. Another drawback, as stated by Baker et al (2007) is the inability of many of these sources to be propagated in a commercial setting or the difficulty in replicating the production of the active molecule in an artificial environment.
Despite the stated drawbacks, there are successful findings of new compounds that lead to further drug development. Examples of such compounds are salinosporamide A yielded from a marine actinomycete Salinispora tropica which inhibited human colon carcinoma HCT116 cells and exhibited extreme potency against several non-small cell lung, central nervous system and breast cancer cell lines (Blunt et al, 2007). Other examples of compounds that are undergoing clinical trials are Bryostatin derived from Bugula neritina in phase II for treating cancer, IPL-550260 derived from a sponge in phase I for inflammation treatment, and Ziconotide derived from mollusk in phase III for treatment of neuropathic diseases (Baker et al, 2007).
2.3.3 Natural Products in Microorganisms
Microorganisms are vastly found in the environment and have structurally diverse bioactive substance. They have been quite an important source in drug discovery process. Penicillin found by Alexander Fleming has become the foundation for development of microbial natural product in drug discovery (Challis, 2008). Microorganisms have largely contributed in development of antibacterial, such as penicillin, cephalosporins and other drug compounds. Nowadays, therapeutic uses of metabolites from microorganisms have been used in several drug classes such as immunosuppressive agents (eg, cyclosporins and rapamycin), cholesterol-lowering agents (eg, lovastatin and mevastatin), antihelmintic agents (eg, ivermectin), antidiabetic agent (acarbose), and anticancer agents (eg, pentostatin, peplomycin, and epirubicin) (Chin et al, 2006).
Among the drugs discovered from microbial sources approved by FDA are rosuvastatin calcium isolated from Penicillium citrinum and P. brevicompactum for management of dyslipidemias; daptomycin isolated from Streptomyces roseosporus for treatment of complicated skin and skin structure infections; and caspofungin acetate from derivation of pneumocandin B0, a fermentation product of Glarea lozoyensis (Chin et al, 2006).
Endophytes, generally can be defined as microorganisms that live in plants. A more inclusive and widely accepted definition is endophytes are microbes that colonize living, internal tissue of plants without causing any immediate, overt negative effects (Strobel and Daisy, 2003). Endophytes, which are a source of natural products, are still poorly exploited. The reason is robots, combinatorial chemistry and molecular biology has replaced the interest of natural products in drug development (Strobel and Daisy, 2003).
However, endophytes have shown as a promising source of new drugs in drug development. Endophytes have large biodiversity as one plant can host a few types of endophytes, and there are numerous plants around the world. Endophytes can be described as having symbiosis with the hosts as they contribute to the health and growth development of the hosts in exchange of some privileges (Tadych and White, 2009). Some endophytes are said to be mutualists that protect the hosts and are responsible of adaptations of plants to stress. Recently endophytes have been the target of new bioactive compounds and second metabolites for drug development.
2.4.1 Anticancer Agents from Endophytes
In anticancer drug development, sources from endophytes have become a success and further research are being done to improve the findings of anticancer agents from the endophytes. A famous compound of anticancer agents that come from this source is paclitaxel. Paclitaxel is found in every yew (Taxus) species (Strobel, 2003). This compound has contributed to the advancement in cancer treatment, especially for breast cancer patients (Luck and Roche, 2002). Endophytes Seimatoantlerium tepuiense and S. nepalense have been reported to produce paclitaxel. However, paclitaxel has some common side effects, which are neutropenia and peripheral neuropathy.
Some new compounds have been prospected to have anticancer activity. Endophyte Pestalotiopsis microspora associated with the endangered tree Torreya taxifolia (Florida torreya) produces anticancer agent, torreyanic acid which is more potent than taxol and induce cell death by apoptosis (Priti et al, 2009). These findings further strengthen the reason why endophytes can be a truly valuable source in discovering new and more enhanced treatment for cancer.