DISCUSSION:
Previously, the immunostimulatory properties of Echinacea were demonstrated by measuring Interleukin-6 production in the J774 macrophage tumor. Echinacea stimulated macrophages to produce large amounts of IL-6, which was above that obtained by the positive control (LPS + IFN). Recently, the ability of Echinacea to activate the J774 macrophages was confirmed by measuring the production of TNF-a using the previously described ELISA. Treatment with Echinacea resulted in a marked increase in production of TNF-a, which is known to be produced in large quantities by activated, but not naïve macrophages. It is these immunostimulatory properties of Echinacea, which are responsible for its effective treatment of the common cold virus, which has been met by its widespread use.

Since differentiation features usually accompany changes in activation status the J774 macrophages tumors were closely examined after Echinacea treatment using light microscopy. It was observed that the morphology of the cells changed from a small round cell, typical of an immature monocyte to a larger, more spread out and mature macrophage. This indicated that Echinacea was stimulating macrophage tumors to become activated and to begin to mature down the path of terminal differentiation. A tumor cell is characteristically blocked at an early stage of its differentiation pathway in which there is usually a strong capacity for cell division and proliferation. The more mature a cell becomes the less it proliferates. Further investigation in the proliferation assay showed that indeed the tumor cells were losing their capacity to proliferate, as their proliferation was inhibited after treatment with Echinacea.

The hypothesis that Echinacea can cause macrophage tumors to mature was tested by evaluating the expression of specific surface markers, which are up regulated as the macrophage becomes more mature. Immunoflouresence staining and confocal microscopy was first employed to directly visualize the expression of markers of differentiation within those cells, which appeared to undergo morphological changes. The increase in MHC Class II and CD11b expression of cells undergoing the morphological changes induced by treatment with Echinacea qualitatively confirmed the differentiation of these tumor cells. In order to quantify the degree of up regulation of the cell surface markers examined by immunofluorescence, we performed flow cytometric analysis to determine if not only the number of cells expressing these markers increased but also if there was a correlating increase in the level of expression on each cell. As expected, treatment of tumors with Echinacea saw both an increase in the fraction of cells expressing CD11b, MHC class I and class II but also an up regulation of the level of expression indicating that not only more cells expressed the markers but there was a higher level of expression when compared to untreated tumours. However, LPS+IFN saw a greater up regulation of class I MHC than Echinacea which is expected given the knowledge that IFN results in a significant up regulation of MHC class I in all cells. These data taken together suggested that these tumors were indeed becoming more differentiated, ceasing to divide and reverting to G0 of the cell cycle and as such assuming a limited life span.

Next, it was examined whether Echinacea resulted in terminal differentiation and subsequent induction of programmed cell death or apoptosis. This was accomplished using the apoptosis assay, which is based on the incorporation of radioactive thymidine within the DNA of proliferating tumor cells during the S or DNA synthesis phase of the cell cycle. Tumors incubated with radioactive thymidine incorporate this into their DNA and after treatment with Echinacea if apoptosis results, there will be ongoing DNA fragmentation and loss of the radioactive thymidine, which is measured as a surrogate for apoptosis. The apoptosis assay showed that tumors incubated with Echinacea showed significantly greater DNA fragmentation or apoptosis when compared to tumors grown in media alone. This is exciting evidence, which clearly demonstrates that Echinacea not only results in differentiation but also, programmed cell death, which is a feature of normal cells. Thus, Echinacea may be suitable as a chemotherapeutic agent in the treatment of cancers such as AML.

From these results it can be concluded that Echinacea activates macrophage tumors, initiates maturation of the tumor and terminally differentiates the tumor, leading to apoptosis. Therefore Echinacea could be used as a cancer treatment on the basis that it causes differentiation of cells and induces apoptosis of the tumor. Cancer is the second leading cause of death in Canada and the United States and is a significant strain on health care worldwide. If Echinacea's effect on cancer cells is confirmed by further studies, it could have a significant impact on the medical community's approach to cancer treatment.

The next step to exploring this possible cancer treatment is to further understand what elements in Echinacea cause an inhibition of proliferation and an increase in apoptosis. More specifically, it is important to determine the cell signaling pathways and genes involved in the anti-proliferative effects of Echinacea. The effects on healthy cells and dosages should be explored both in vitro and in vivo in order to better evaluate its potential use as a treatment of tumors.