Hypericum perforatum Extracts and Hypericin Treatment of a Mouse Mammary Cancer Cell Line Induces Growth Inhibition in a Dose Dependent Manner

Hypericum perforatum, commonly known as St. John’s Wort, has been found to exhibit many medicinal, especially anti-depressant, properties. Hypericin is thought to be the main chemical constituent responsible for H. perforatum’s medicinal properties. We report here the ability of H. perforatum and hypericin to inhibit the growth of mouse mammary cancer CRL2539 cells. H. perforatum, at concentrations of 0.4% and 0.8%, significantly (P<0.05) inhibited cell growth in a concentration dependent manner. Hypericin (purity 80-90%) at a concentration of 0.001% also significantly inhibited cell growth but not to the extent which the H. perforatum extracts did. In addition, H. perforatum at a concentration of 0.8% inhibited cell growth significantly more than H. perforatum at a concentration of 0.4%. Our study shows a promising therapeutic strategy in using the whole H. perforatum extract as its own form of treatment to effectively slow the growth rate of cancer cells, and potentially overcome the negative side effects associated with current forms of cancer treatment. Introduction Hypericum perforatum is a yellow flowering plant naturally found in various locations around the world including West Asia, Europe, and North Africa. Hypericum perforatum has been studied for its effects in treating depression and attention deficit hyperactivity disorder1. Cancer prevention research is one of the new areas that Hypericum perforatum and its main chemical constituents are being applied to. Research has shown that the most significant compounds in Hypericum perforatum for cancer prevention are hypercerin and hyperforin2. Hypericin is used in photodynamic therapy (PDT) as a form of cancer treatment. PDT requires a photosensitizing agent (photosensitizer) and visible light of a wavelength that correlates with the absorption spectrum of the drug. Alone the light and photosensitizer have no therapeutic effect, but when combined produce cytotoxic products which trigger irreversible tumor destruction and cell damage. Out of 36 species of Hypericum, 27 held hypericin, the most common being Hypercium perforatum3. The cell type in which hypericin is applied dictates the killing efficacy and the cellular distribution of the drug. In colon carcinoma CAco-2 cells for example, hypericin is found to accumulate in the nuclear and plasma membranes. Hypericin mainly targets cell membranes and can affect critical mitochondrial functions in a photodependant manner. Although hypericin does not gather in mitochondria, hypericin’s photodynamic action Materials and Methods CRL2539 mouse mammary cancer cells (American Type Culture Collection) were seeded at concentrations of 1.0 x 104/25cm2. Hypericum perforatum Extracts and Hypericin Treatment of a Mouse Mammary Cancer Cell Line Induces Growth Inhibition in a Dose Dependent Manner Ashley Ferguson1*, Caitlin Morris1*, and Jackie Curley2 Student1, Teacher2: The Loudoun County Academy of Science, Sterling VA *these authors contributed equally. *correspondence: [email protected]; [email protected] primarily targets these cell sites as shown by the impairments in mitochondria bioenergetics when hypericin is present and combined with a visible light wavelength3. Another form of cancer treatment that involves the use of hypericin is catalytic therapy (CT). CT is a cancer treatment that involves the use of substrate molecules and a catalyst to generate reactive oxygen species (ROS). Hypericin from Hypericum perforatum as an active photosensitizer assists in ROS generation in response to light. Hypericum perforatum alone has little effect on cell life but when an activation mixture, made up of ascorbic acid, is added, it drastically increases early apoptosis in cells4. These researchers could not find studies delineating the effects of the whole extract as opposed to its component compounds. Although hypericin from Hypericum perforatum has had positive effects in cancer treatments so far, when combined with other drugs for in vivo treatment it can cause dangerous side effects such as reduced plasma levels of antiretroviral agents, which increases patients’ risk for disease progression2. Also, when used as a photosensitizer in photodynamic and catalytic therapy, an increase of dosage of hypericin results in a shift from apoptotic to necrotic death resulting in a harmful inflammatory response in the patient3. Our study tests the effects of a whole extract of Hypericum perforatum and one of its chemical constituents, hypericin, on the growth of a mouse mammary cancer cell line. The first hypothesis is that if Hypericum perforatum and hypericin extracts are applied to a mouse mammary cancer cell line, then the cancer cells exposed to the extracts will grow at a slower rate than the control cells. The second hypothesis is that if Hypericum perforatum and hypericin extracts are applied to a mouse mammary cancer cell line, they will the slow the cell growth at an equal rate. The third hypothesis is that if Hypericum perforatum is added in greater concentrations to the mouse mammary cancer cells, then the cells will grow at an increasingly slower rate. Our study is important is because although hypericin has been used before in cancer such as PDT or CT, there are many negative side effects associated with current treatments such as increased risk for disease progression and induction of an inflammatory response. If we could find the optimal dosage of hypericin and Hypericum perforatum extract, it could be used on its own as a separate form of cancer treatment. 14 The Journal of Experimental Secondary Science January 2012 Volume 1 Issue 3 Cancer inhibitory activity of liquid glycerol based Hypericum perforatum extract (0.3% hypericin) (Puritan’s Pride Incorporated) and hypericin (Planta Analytica) were determined by exposing cells to their various concentrations for 96 – 264 hrs. or until control cells reached confluency. Each set of trials were run with three 25 ml control flasks, three 25 ml 0.4% Hypericum perforatum flasks, and three 25 ml 0.8% Hypericum perforatum flasks or three 25 ml 0.001% hypericin flasks. Seven mL of media were needed for each flask, so media was made in sets of 21 mL with dilutions as follows. 21 mL of DMEM with 10% FBS were made for the control cells. 0.084 mL of Hypericum perforatum and 20.916 mL of DMEM with 10% FBS were made for the 0.4% Hypericum perforatum flasks of cells. 0.168 mL of Hypericum perforatum and 20.832 mL of DMEM with 10% FBS were made for the 0.8% Hypericum perforatum flasks of cells. 0.021g of hypericin was dissolved in 1mL of ethanol for the 0.001% hypericin flasks of cells. This was added to 20mL DMEM. 1mL of ethanol was added to 20mL of DMEM for the ethanol flasks of cells. Cells were cultured in a 5% CO2, 95% air, fully humidified incubator at 37°C. Control CRL2539 cells were cultured in DMEM containing 10% FBS. Experimental CRL2539 cells were cultured in DMEM containing 10% FBS and 0.4% Hypericum perforatum, 0.8% Hypericum perforatum, or 0.001% hypericin. Cell growth was estimated at periods of 24 – 48 hrs. using pictures from the inverted microscope. Three pictures were taken at randomly dispersed locations throughout each flask. The cells in each picture were counted and averaged so that the number of cells per mm2 was found. This number was multiplied by the surface area of the flask, 1763 mm2, to find the average number of cells in each flask at a certain time. At the end of each trial we found the final cells counts in each flask using a hemocytometer. We performed a hemocytometer count per flask three times and average the number of cells counted to ensure an accurate representation. Statistical evaluation of the results was performed with the Student’s t-test using the Microsoft Excel software. Probability values equal or less than equal to 0.05 were considered significant. Normal distribution graphs were constructed based t-test values using Fathom Dynamic Data software. Ashley Ferguson, Caitlin Morris, and Jackie Curley Page 2 of 5