A high throughput anti-cancer drug leads screening method has been successfully established employing the cell line we reported in 2001. The method is based on the principle of fluorescence resonance energy transfer (FRET) and fluorescent protein variants of GFP. This cell-based method has advantages over the animal model screening and biochemistry-based screening. Compared to animal model screening, this cell-based method will save a lot of time and money in screening. At the same time, the cell-based screening method considers the cell context. The screening results will reflect interaction between compounds and cells or compounds and proteins in the cells compared to biochemistry-based screening....[ Read more ]

A high throughput anti-cancer drug leads screening method has been successfully established employing the cell line we reported in 2001. The method is based on the principle of fluorescence resonance energy transfer (FRET) and fluorescent protein variants of GFP. This cell-based method has advantages over the animal model screening and biochemistry-based screening. Compared to animal model screening, this cell-based method will save a lot of time and money in screening. At the same time, the cell-based screening method considers the cell context. The screening results will reflect interaction between compounds and cells or compounds and proteins in the cells compared to biochemistry-based screening.

The YFP/CFP emission ratio (Y/C emission ratio) has been used to judge the screening result. The distinguished advantage of this cell-based screening method is that the Y/C emission ratio is only response to apoptotic cell death but not necrotic cell death. Thus, the method can distinguish apoptotic cell death from necrotic cell death. The compounds, which can significantly induce cell apoptosis, will be picked out from the compound library after screening.

Optimization has been done for the screening procedure to enable it high throughput and low cost. In the process of optimization, it is found that medium only can be used as background. At the same time, the initial cell confluence and fluorescent plate reader parameters were also optimized. By now, a standard protocol has been established. Five anti-cancer drugs have been tested by this method. The screening results showed that the screening method is an excellent screening method. In addition, a new method to determine screening threshold has been developed based on the relation between Y/C emission ratio and cell viability. The screening threshold was set to 3 for our screening setup, which means that the compound that can induce Y/C emission decreases below the 3, will be considered as effective compound.

Several small scale screening projects have been done. The screened compounds belong to two compound families: tanshinone compound family which were extracted from Salvia miltiorrhiza Bunge and podophyllotoxin compound family which were extracted from Podophyllum emodi Wall., which are all herbal extractions or their derivatives. In podophyllotoxin family, the deoxypodophyllotoxin (DP1) is the most potent anti-cancer compound. In tanshinone compound family, it is found that tanshinone IIA (TIIA) is the most potent compound on killing cancer cell. This result is consistent with reference.

Although tanshinone IIA is potent in killing cancer cells, its solubility in water is not good. With chemical modification, a new compound called diacetyltanshinone IIA (ATA) was got. The solubility of ATA is improved compared to TIIA. The toxicity and mechanism studies were also carried out on ATA. In vitro toxicity assay results show that this compound has inhibition and killing effects on various cancer cell lines. And ATA anti-cancer effect is better than its parental compounds TIIA on some cancer cell lines. At the same time, the mechanism studies showed that ATA can induce cell apoptosis through mitochondrion-dependent pathway. The further investigation revealed that reactive oxygen species (ROS) generation and intracellular Ca²⁺ level decrease may play an important role in the process of ATA induced apoptosis.

Establishment of methods and preliminary screening results demonstrates that our screening platform is a powerful and reliable tool for anti-cancer drug leads discovery. And ATA is a promising anti-cancer compound.