BACKGROUND

Cancer is a large class of malignant diseases, 14 million new cases arising and causing over 8 million deaths per year. Which is the second most lethal disease in the world._[1]

Anti-microtubule agents , one of the most efficient medicines to treat many cancers including breast cancer、ovarian cancer and so on , are plant-derived chemicals that block cell mitosis by interfering with microtubule function . Microtubule is an important cellular structure composed of two proteins; α-tubulin and β-tubulin. They are hollow rod shaped structures that are required for cell division, among other cellular functions. _[2] Microtubules are dynamic structures, which means that they are permanently in a state of assembly and disassembly. And the anti-microtubule agents can inhibit the dynamic balance of microtubule, leading to the termination of cell mitosis and inducing the apoptosis of tumor cells.

Anti-microtubule agents can be divided into two types : one inhibit assembly , such as vinca alkaloids、colchicine、podophyllotoxin and so on . The other inhibit disassembly , including taxanes、epothilones etc. Taxanes is the most famous among them.

Paclitaxel was discovered in 1962, isolated from the bark of the Pacific yew, Taxus brevifolia, thus its name “paclitaxel”. After only a brief period, the taxanes have demonstrated a unique ability to palliate the symptoms of many types of advanced cancers, including carcinoma of the ovary, lung, head and neck, bladder, and esophagus. Due to its good efficacy and slight side effect, the taxane class of anti-microtubule anticancer agents is perhaps the most important addition to the chemotherapeutic armamentarium against cancer over the past several decades.

Paclitaxel and other anti-microtubule medicines make a great success, which inspires many factories to extract these compounds, leading more and more famers to cultivate plants containing these compounds widely. Moreover, there are more and more scientists devote to study them. However, under this circumstance, many problems have alsobeen caused.

As to our project this year, we modified the homo sapiens tubulin alpha 1a , connected it with luciferase report gene’s N terminal or C terminal , and we put the modified α-tubulin and β-tubulin into E-coli to reduce them . Then we will get a kit containing the tubulins and buffer which has the appropriate condition verified by experiments. We call the kit “taxolight”, and it can do these things:

1. drug screen

Anti-cancer agents especially paclitaxel have showed their magnificent power in clinical application, but also are a little unsatisfactory. We still need to look for new drugs that more effective.

The existing method to screen anti-microtubule agents needs purifying tubulins of mammalian brains. It relies on the features of tubulins that the solution turbid will increase when then polymerize in vitro under 37℃ centigrade. So using this method, we can get a polymerization curve shaped sigmoid formed by the liquid OD value to the soaking time, correspondingly, we can also get a de-polymerization curve when putting the tubulins into ice. When adding different anti-microtubule agents, polymerization of "S" type curve or pour de-polymerization of "S" type curve has different effects, and we can determine the role of the drug according to the change of curve. The defects of this method are as follows:

1. The operation of exacting and purifying tubulin from animal brain is very complicated, and fresh brain tissues are needed, so that the experiment must be done within an hour after killing the animal. At the same time, the price of reagents in this experiment is expensive. The experiment period is long which takes 3 days to purify the tubulins.
2. The wave length of measuring the OD is 350nm, which is between ultraviolet light and visible light. This leads to a huge deviation, so the instrument requirement is strict. And because the 350nm wave length lies in the UV light region, quartz containers are needed, which costs a lot.

Our project avoids these drawbacks, and provides a new idea on the drug screen of anti-microtubule agents: using our kit, add the two kinds of quantitative modified α-tubulins withβ-tubulins and buffer, and put the quantitative sample, and then measure its fluorescence intensity. Paclitaxel can be set as a standard, and we can compare the new medicine with paclitaxel by comparing the fluorescence intensity. In this way, the primary invitro screening of compounds that can influence microtubules can be carried out using our kit, to further research and development of new anti-microtubule agents.

2. Detect the existence

HPLC/RP-HPLC is one of the most common method for detecting paclitaxel now. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.

The shortcoming is that the progress need a long time, which is very unfavorable to the studies in laboratory. For example, in a laboratory which producing paclitaxel from fungus, detecting the concentration of the product may delay experiment process if there were no paclitaxel at all. So we need to develop an effective method to rapidly detect whether taxanes exist or not before measuring concentration, which our kit can achieve in order to accelerate research progresses.

3. Detect the concentration

On the basis of successfully achieving the former functions, we still want to improve our kit, so that it can be more powerful. Our plan is: we can establish an intensity – concentration database of a certain medicine (e.g. paclitaxel), then we can use our “taxolight” to determine the concentration of this certain medicine conveniently.

There is a limiting condition when using our kit to determine the concentration that it must be ensured that the sample solution doesn’t have other anti-microtubule agents. Nevertheless, it still has a high value in use. For example, famers who plant taxus can use our kit to detect the concentration of taxanes in there plants. Taxus need two years to harvest. And different species of yew has different paclitaxel content, some even don’t contain paclitaxel. Paclitaxel content is also closely related to the planting technique and the environment. This summer, we went to Yunnan, where is famous for abundant Taxus. And at there, we learned a terrible thing: In 2001, many farmland in Maguan county are planted with taxus , but due to the lack of planting techniques, most of the taxus can’t be used . That caused a series bad consequences. If our kit can be used by famers, they will be able to detect conveniently and notice it early, thus the tragedy will be effectively prevented.

As a huge breakthrough in cancer treatment, anti-microtubule drugs are of great significance. Our project helps shed light on the standard screening in drug developmental phase. Moreover, with our further optimization, we hope that we can achieve a more accurate level in the quantification of taxol or other anti-microtubule drugs compared to conventional testing methods.