1. Screening Natural Product Extracts and Pure Compounds for their Effects on the Anti-Tumor Response of Natural Killer Cells

The overall goal of this project is to discover agents which stimulate and/or regulate the in vitro antitumor activity of NK cells. Since natural products have been shown to contain a diverse universe of chemotypes with potent drug-like activities, then screening extracts and their purified constituents obtained from various microbes and plants will yield compounds whose biological activities will stimulate and/or regulate the in vitro activity of NK cells and therefore, result in an enhanced anti-tumor effect. NK-92M1 is an interleukin-2 (IL-2) independent natural killer cell line which retains many of the properties of freshly isolated NK cells, including cytotoxicity towards human tumor cell targets. The tumor cell target, the K-562 human leukemia line, was derived originally from a 53 year-old female CML patient in blast crisis Our experiments are designed to measure NK-cell activity cultured in the presence or absence of test extracts and/or compounds using 1) flow cytometry and 2) microtiter based lactate dehydrogenase assays. Extracts and/or pure compounds are obtained from research collaborators, which include both academia and industry/biotech. Those extracts which are found to be stimulatory to NK cell activity undergo chemical extraction and purification by our collaborators, and the resulting fractions and pure compounds are further assayed for NK cell activity using flow cytometry. Those compounds which enhance or modulate NK cell activity are further identified as to structure and/or chemotype by our collaborators.

Our current efforts include the investigation and optimization of flow cytometric analyses of NK cell activity, using a novel combination of fluroescent and non-fluorescent dyes to distinguish NK cell mediated target cell killing.

 2. Discovery of Anti-Tumor Agents from Natural Products. The focus of this project is the discovery of new anti-mitotic agents whose mechanism of action includes subversion of spontaneous developing drug resistance in tumor cell populations. Natural product extracts and pure compounds are screened for anti-proliferative activities using two human non-small cell adenocarcinoma cell lines, A549 and the multi-drug resistant NCI-H1975. Tumor cell assays employed in the research include microtiter colorimetric (MTT) based assays to measure proliferative capacity. Extracts and pure compounds which exhibit potent anti-proliferative activity in both sensitive and drug resistant cell lines are further analyzed for mechanism of action using cell cycle analyses and flow cytometry.

A549 Adenocarcinoma Cells

We are currently screening terrestrial and marine derived extracts and pure compounds in collaboration with several investigators from both academia and industry. Several extracts have demonstrated potent cytoxic activities towards both lung cell lines and we are following up on determining their mechanism(s) of action.

3.  The Flow Cytometry Core Facility.  This is a laboratory resource available for LECOM researchers whose investigations require cellular phenotypic and functional analyses using flow cytometric technology. The facility houses a Beckman Coulter EPICS Altra flow cytometer/cell sorter.  This is a three laser configured instrument consisting of a Coherent Enterprise II water cooled  Argon/UV laser, Melles Griot air cooled HeNe laser, and an air cooled argon laser, capable of exciting a wide range of fluorochromes such as FITC, PE, PerCP, ECD, PE-Cy5, APC and DNA dyes such as DAPI, Hoechst and PI.  The instrument is configured for up to 5 color simultaneous analyses with gated amplifier.  If cell sorting is desired, sort purities of  >95%, with an event analysis rate of up to 10,000 events per second can be obtained with this instrument. Analyses of flow cytometric data is accomplished using EXPO32 software in a workstation environment.

A wide variety of cell types have been analyzed and/or sorted using this instrument, including human, mouse and rat tumor cell lines, primary cell cultures from multiple mammalian species, bacteria, algae and fungi. Some of the techniques routinely employed in his laboratory include extracellular cell surface and intracellular biochemistry marker analyses, cell cycle analyses, cell ploidy, apoptosis, necrosis, calcium influx, function of Pgp multi-drug resistance pump and natural killer (NK) cell activity.