Cleavage is the process in development which creates a multicellular organism through rapid cell divisions. During the cleavage period of development, cell divisions rates are more rapid than any other time in the life of the individual (Gilbert, 2003). Cleavage is actually the result of two coordinated cyclical processes. The first of these is karyokinesis, which is the mitotic division of the nucleus. The mechanical agent of karyokinesis is the mitotic spindle, with its microtubules composed of tubulin. The second process is cytokinesis which is the division of the cell. The mechanical agent of cytokinesis is a contractile ring of microfilaments made of actin (Gilbert, 2003).
Karyokinesis is an essential part of development because microtubules are responsible for various cell movements. Microtubule functions include the beating of cilia and flagella, the transport of membrane vesicles in the cytoplasm, and, in some protists, the capture of prey by spiny extensions of the surface membrane. These movements result from the polymerization and depolymerization of microtubules or the actions of microtubule motor proteins (Aaronson, et al., 2003). Some other cell movements, such as the alignment and separation of chromosomes during meiosis and mitosis, involve both processes. Microtubules also direct the migration of nerve-cell axons by promoting the extension of the neuronal growth cone. The inhibition of microtubules is the target of many cancer fighting compounds. Research has suggested that the inhibition of microtubules results in mitotic arrest and prevents the cell from dividing and later results in cell death. Thus, the inhibition of microtubules proves be one of the key targets for chemotherapeutic agents.
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Microtubules are intrinsically dynamic polymers, undergoing two kinds of dynamic behavior: dynamic instability and treadmilling. In dynamic instability, microtubule ends stochastically switch between episodes of prolonged growing and shortening (Mitchison and Kirschner, 1984). One microtubule end, the plus end, shows more dynamic behavior than the opposite end, the minus end. The other form of dynamic behavior, treadmilling, consists of net growing at microtubule plus ends and net shortening at minus ends (Margolis and Wilson, 1978). Microtubule dynamics are important to many functions in cells, the most dramatic of which is mitosis. When cells enter mitosis, the interphase cytoskeletal microtubule array is disassembled and a bipolar spindle is assembled. The kinetochore is where the spindle microtubule and the chromosomes attach and contribute to chromosome alignment and subsequent segregation at anaphase. Microtubule dynamics are relatively slow in interphase cells, but increase 10-to 100-fold at mitosis (Saxton, 1984). Both extensive dynamic instability and treadmilling occur in mitotic spindles, and the rapid dynamics of spindle microtubules play a critical role in the intricate movements of the chromosomes.
Inhibition of Microtubules
Assembly of the metaphase spindle happens in two steps, add-on of spindle microtubules to the poles and capture of chromosomes by kinetochore microtubules. At the opposite end of the spindle microtubules, there occurs speedy rise and fall in their length which captures chromosomes during prophase, this occurs as the nuclear membrane starts breaking down (Lin, 2003). By quickly lengthening and shortening at its (+) end, the dynamic microtubule is able to capture the chromosome-rich environment. The microtubule end can contact a kinetochore, scoring a "bull's-eye" (Aaronson, et al., 2003). More commonly, the kinetochore is able to attach at the side of the microtubule and move along towards the (+) end that involves kinesins on the kinetochore. The chromosome is able to attach to the (+) end of the spindle either directly or through side by sliding process, the kinetochore "caps" the (+) end of the microtubule. Therefore each sister chromatid in a chromosome is captured by microtubules arising from the nearest spindle poles. Each kinetochore also becomes attached to additional microtubules as mitosis progresses toward metaphase (Aaronson, et al., 2003).
Microtubule targeting agents represent a diverse group of antimitotic drugs that fall into three categories based upon where they bind to the microtubules. MTAs such as paclitaxel, which bind the taxane domain on microtubules, increase the stability of the microtubule lattice, resulting in microtubule bundling (Dunn 2005). MTAs such as 2-methoxyestradiol (2ME2), which binds the colchicine domain, and vinblastine, which binds the Vinca domain, destabilize microtubules, increasing the amount of soluble tubulin. MTAs that either stabilize microtubules by binding to the taxane domain, or destabilize microtubules by binding to the colchicine or Vinca domains, result in blocking the cell cycle, inhibiting cell growth and inducing cell death (Dunn 2005).
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Eukaryotic cells arrest in metaphase when microtubule polymerization is disrupted. The inhibition of microtubules results in the prevention of mitosis and causes the formation of an incomplete metaphase plate of chromosomes and alters the arrangement of spindle microtubules (Lin, 2003). This type of microtubule inhibition proves useful when one is trying to prevent cell division. One of the most successful inhibitors of microtubules is the Taxane family.
In essence, the taxanes alter the tubulin rate dissociation constants at both ends of the microtubules, thereby stabilizing microtubules against depolymerization. At substoichiometric concentrations, the taxanes suppress microtubule dynamics without appreciably increasing the rate of formation of polymerized tubulin (Aaronson, et al., 2003). The taxanes also induce tubulin polymerization and increase microtubule mass, which occur at stoichiometric binding and submicromolar concentrations that are readily achieved in the clinic. The microtubules of Taxane-treated cells are extraordinarily stable, resisting depolymerization by cold, calcium ions, dilution, GTP, and other antimicrotubule agents. These actions result in the suppression of treadmilling and dynamic instability which are essential for normal microtubule dynamics during both mitotic and nonmitotic phases of the cell cycle. Both stoichiometric and substoichiometric binding of the taxanes inhibit the proliferation of cells, principally by inducing a sustained mitotic block at the metaphase/anaphase boundary; however, morphologic findings, such as the formation of microtubule bundles during the mitotic phases of the cell cycle, suggest that the interphase microtubules in nonproliferating cells may also be affected (Aaronson, et al., 2003).
The History of Taxol
In the 1960's, the National Cancer Institute (NCI) collected plant specimens from around the world with the hope of discovering new medicinal plant compounds. Taxol, which is derived from the Taxane family, was discovered at Research Triangle Institute (RTI) in 1967 by Dr. Monroe E. Wall and Dr. Mansukh Wani. The compound was isolated from the bark of the Pacific Yew tree (Taxus brevifolia), a slow-growing tree found in the virgin rain forests of the Pacific Northwest United States (Metts, 2001). Extractions from the yew bark were taken in very low concentration and were sent to the laboratories of Wall and Wani for further analysis. The Pacific Yew tree (Taxus brevifolia) was the first plant species to demonstrate anti-cancer properties. Extensive research done by a team at The University of California concluded that this is because Taxol binds reversibly to microtubules and at equivalent intracellular concentrations, suppresses the rates of growing and shortening of individual microtubules in both types of tumor cells. Taxol was found to inhibit cell proliferation and block mitosis by preventing progression from metaphase to anaphase. Further experimentation strongly indicates that the mechanism of inhibition of mitosis by Taxol is due to inhibition of microtubule dynamics. Thus, Taxol is an important new cancer chemotherapeutic agent that is effective in the treatment of many types of cancer, including ovarian cancer which has proven to be one of the most susceptible cancers to Taxol treatment.
Fortunately, the occurrence rate for ovarian cancer is relatively low. However, cancer of the ovary is the fourth most common cause of cancer-related death among women. Survival is excellent within the early stages (with surgical removal being the treatment of choice) and poor within advanced stages (with chemotherapy treatment). Chemotherapy in advanced ovarian cancers has improved the 5-year survival rate from 20%-30% (Metts, 2001). It can improve or possibly cure some patients with the advanced disease. However, approximately 20% of ovarian cancers, depending on histologic type, do not respond to any form of chemotherapy. Therefore, Taxol became a new alternative method for treating ovarian cancer. The use of Taxol in ovarian cancer patients who failed initial or subsequent chemotherapy for cancerous diseases resulted in a response rate of 16.2% to 30% with a median survival of 8.1 and 11.5 months for the Phase I and Phase II clinical trials, respectively (Metts, 2001).
The Affect of Taxol on Ovarian Cancer Cells
Taxol, more properly known as paucities, interferes with the normal function of microtubule growth. In contrast to other cancer drugs that cause the deterioration of microtubules, Taxol helps stabilize their structure and function. This destroys the cells ability to use its cytoskeleton in a flexible manner. Specifically, Taxol binds to the proteins of microtubules and locks them in place (Powledge, 1998). The resulting microtubule, now containing Taxol, does not have the ability to disassemble. This adversely affects cell function because the shortening and lengthening of microtubules is necessary for their function as a transportation highway for the cell.
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One common characteristic of most cancer cells is their rapid rate of cell division. In order to accommodate this, the cytoskeleton of a cell undergoes extensive restructuring. The precise mechanism by which mitotic arrest is linked to cell death has not been determined, but the taxanes do interact with numerous regulatory proteins and oncogenes that bind to the mitotic apparatus (Aaronson, et al., 2003). The taxanes induce either apoptosis or programmed cell death through activation of caspases 3 and 8 or a process of "slow death" by means that neither trigger caspase activation nor use mechanisms associated with apoptosis (Aaronson, et al., 2003). Following taxane treatment, even at substoichiometric concentrations that do not increase microtubule mass, cells exit from mitosis but do not continue to proliferate. Instead, substantial DNA fragmentation, indicative of apoptosis, is noted and cell death occurs in 2 to 3 days.
Taxol is an effective treatment for aggressive cancers because it prevents the process of cell division by destroying its flexibility. Taxol effectiveness is also due to the fact that it is highly lipophilic and insoluble in water. Other cells are also affected, but since cancer cells divide much faster than non-cancerous cells, they are far more susceptible to Taxol treatment (Brincat, et al., 2002).
Microtubules, which are key components of the scaffolding (cytoskeleton) of cells, are long, tube-shaped proteins (tubulin) required for many cell functions, including cell division (mitosis), cell shape maintenance, intracellular transport, extracellular secretion, cell signaling and cell motility (Dunn 2005). They are composed of a- beta tubulin heterodimers, which polymerize and depolymerize to lengthen and shorten the microtubules. Microtubules are highly dynamic, with rapid changes occurring in microtubule growth and length, particularly during cell division (Dunn 2005).
Cancer cells proliferate more rapidly than cells in normal tissue and, due to their rapid proliferation, cancer cells can be destroyed by drugs known as microtubule targeting agents (MTAs). The MTAs bind to tubulin in microtubules and prevent cancer cell proliferation by interfering with the microtubule formation required for cell division. This interference blocks the cell cycle sequence, leading to apoptosis (Dunn 2005).
The exploration of Taxol and the Taxane family will never be complete. Many medical groups will continue to search for ways of synthesizing and improving the anti-cancer drugs. Even though drugs in the past have had progress with treating cancer, Taxol is the most promising anti-cancer agent to be discovered in the last 20 years. It has opened a door of endless possibilities for the medical world and has given hope that cancer can indeed be successfully treated and hopefully, one day be eliminated.
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Taxol: A Microtubule Inhibitor