Johnathon Kreider and Faculty Mentor: Brad Geary, Plant and Wildlife Science

Introduction
Drug testing is one of the most important activities of the Life Sciences. As biotechnological resources like genetically modified mice or artificial skin become more commonplace and accessible to drug researchers, testing has become more efficient. One of the reoccurring problems, though, is that for new drugs to be tested they must first be found. Research has shown that plants and fungi are the best places to look: over 70 percent of drugs that have been introduced in the last quarter century have been derived from natural resources (Newman& Cragg 2012). Notable examples such as Taxol from the Yew tree, Aspirin from the Willow tree, Codeine from the Opium poppy, and Digitalis from the European foxglove prove that it is productive and beneficial to consider natural resources for drug discovery.
Drug discovery to combat cancer is a current “hot spot” of research, and because many treatments for various types of cancer have come from natural sources, our study aimed at doing the same. Some studies had shown that an Asian/European fungus, Phellinus igniarius, had potential to stop cancerous cells from growing (Song, Lin, Yang, & Hu 2008). The overall goal of this study was to explore the biological effects of the Alaskan variety of Phellinus igniarius by testing it to determine activity against cancer cells to prompt further research in the fungi.

Methodology
Fruiting bodies of P. igniarius were collected from the Kenai Peninsula of Alaska, dried, and ground into powder. After boiling in distilled water for 3 hours, the extract was mixed with two volumes worth of cold (-20°C) 95% ethanol and centrifuged. Both pellet and supernatant were collected and briefly suspended in distilled water, then freeze-dried. For the cancer assay, MDA-MB-231 breast tissue cells were grown in flasks, trypsinized, and separated onto 96-well plates with PBS media. After incubation, the cells were treated with various concentrations of the pellet and supernatant extracts of P. igniarius as shown in figures 1 and 2. After incubation with the treatments for 48 hours, the 96-well plates containing the cells were treated with Alamar Blue reagent and incubated at 37 ͦ C for 6 hours. Cell viability was then measured via FLUOstar OPTIMA plate reader to determine the percent of living cells.

Results
It was hypothesized that either the supernatant or the pellet would have a bioactive compound, not both, but after following the standard procedures and running the assay, both the pellet and supernatant of the P. igniarius extract were bioactive against the cancer cells in similar concentrations. We found that the KD50 for the cancer cells treated with the pellet extract was 1.24mg/ml (See Fig. 1, R2= .9787), and the KD50 for the cancer cells treated with the supernatant extract was 1.51mg/ml (See Fig 2, R2= .9922). Controls were included, and as expected, had no effect on the cancer cells. These tests were run three times to ensure the accuracy of the results.

Discussion
Further research will be necessary to see if the extracts are active against multiple cell lines. Now that the extract has been shown to have anticancer effects, however, our next objective is to identify exactly which secondary metabolites in the extract are bioactive against the cancerous cells. Once purification is completed, the extracts will be compared against other cancer treatments and tested in animal models to determine if they would be effective potential cancer treatments.

Conclusion
This study acts as a microcosm of other future studies. While it is necessary to do more research to determine the effects of the P. igniarius extract, efforts geared towards finding new drugs need to be enlarged. Only 2% of the available plants and fungi in the world have been studied for medicinal effects, producing most of the drugs that are commonly used throughout the world today (Farnsworth, Biodiversity). Various cancer drugs, especially those targeting major epithelial cancers, need to be researched with vigor. Advances in modern biotechnology have provided all the tools needed to test compounds in a cost-effective way. The more fungi and plants are tested for bioactivity, the more drugs will be found.