No abstract

Our laboratory is interested in searching for a new plant‐based therapeutics to treat ovarian cancer. We are interested in studying anti‐cancer effects of KY grown hemp as a potential candidate drug. Marijuana and hemp belong to the same genus and species. However, they are different in cannabidiol (CBD) and tetrahydrocannabinol (THC) content. While both CBD and THC are therapeutically beneficial, THC consumption leads to dependence forming habits. In contrast, hemp is harmless and non‐addictive. Major objective of this study is to investigate whether KY hemp extract can modulate the metastasis of ovarian cancer. Cell migration and invasion are two important indicators of cancer metastasis. Although CBD and THC‐induced attenuation of cancer cell migration and invasion have been reported in several cell lines including cervical cancer and breast cancer cells, KY hemp extract‐induced anti‐cancer effects are yet to be discovered. We are the first to investigate KY hemp‐induced modulation of ovarian cancer cell (OCC) metastasis.MethodsBased on the manufacturer's protocol cell migration and invasion assays were conducted using CytoSelect 96‐well cell migration and invasion assay kits (Cell Biolabs). Migration and Invasion assays were based on distinguishing migratory and invasive cells from non‐migratory and non‐invasive cells. Migratory cells should be able to move towards the chemoattractant. Invasive cells needed to degrade the proteins in the dried basement membrane matrix, which is coated in the upper surface of the insert membrane. Two different epithelial OCCs, which are A2780 and Mes‐OV, were utilized in this study. Following overnight serum starvation, OCCs were lifted using Trypsin‐EDTA, and a cell suspension containing 80,000 cells per well were placed in the upper chamber of either the cell migration or the cell invasion plate. Added to the respective cell suspensions were positive controls (80 μM Doxorubicin and Cisplatin), the no hemp negative control (vehicle treated), and different concentrations of KY hemp extracts that contain 8.5–335 μM CBD. Fetal bovine serum (15%) was added to the bottom chamber of each well as the chemoattractant. After 24 hour incubation, migratory and invasive cells were detached from the opposite side of the membrane (the side facing the bottom chamber), lysed, tagged with CyQuant fluorescent dye, and quantified spectrophotometrically. Our results indicate that KY hemp extract will attenuate OCC migration in a dose dependent manner in both cell lines. In A2780 OCCs, this attenuation was significant at all concentrations tested. A2780 cells treated with 12–15 μg/ml hemp extract that contain 2.5–3.2 μM CBD, caused decrease in cell migration comparable to Cisplatin. Based on the data here we conclude that KY hemp has significant anti‐metastatic properties against ovarian cancer.Support or Funding InformationFunding was provided by Sullivan University System via a faculty development grant awarded to Dr. Sumanasekera. Grant Number: RG_1_PS_2017_04This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ovarian cancer is one of the most aggressive gynecological cancers (Matz et al., 2017) and the fifth leading cause of cancer‐related deaths among women (Siegel et al., 2015; American Cancer Society, 2019). The current treatment options include Cisplatin and Liposomal Doxorubicin. However, due to limited efficacy and unwanted side effects of the existing therapeutics, it is important to look for alternative therapies. Due to growing interest in medicinal treatments with natural substances, we began a search for natural products with anti‐cancer activity. Most of the existing literature is about the use of medical cannabis or Dronabinol for cancer‐related side effects management. Some other studies are beginning to emerge on the anti‐cancer effects of certain cannabinoids or mixtures of cannabinoids on few cancers. However, no one has reported the anti‐cancer properties on Hemp Extract (HE) that we utilized. The objective of this study is to investigate whether HE has anti‐cancer properties against ovarian cancer cells (OCCs) while being safe to normal ovary epithelial cells (NHOECs). Our specific aims were to investigate 1) Dose response relationship and half maximal effective concentration (EC 50) for HE; 2) HE's ability to specifically decrease ovarian cancer cell (OCC) viability; 3) Specifically cause cytotoxicity to OCCs; 4) Specifically decrease OCC death; and 5) Decrease metastasis of OCCs. Our methods include preparation and purification of HE, cannabinoid amount and molarity calculations, cell culture, HE Treatments, EC 50 calculations, and multiple cell‐based assays / detection methods including cell viability assays (MTT, CALCEIN), cell death detection (ETHD‐1), apoptosis assays (CASPASE), phase contrast microscopy, cytotoxicity assays (LDH), and the cell migration assay. The HE was produced using supercritical carbon dioxide extraction method and given to the laboratory. This extract was made from the flowers, stalks, and leaves of the seedless hemp plants. HE stocks and different working concentrations were made and purified in the laboratory. We have treated cultured OCCs (A2780 and MES‐OV) and NHOECs with different concentrations of HE for 24 hours. Cannabinoid amounts and molarity present in each hemp treatment concentration were calculated. Dose repose relationships were established, and EC 50 was calculated for A2780 OCCs treated with HE. Several cell‐based assays were conducted to fulfill each of the specific aims. We found that the EC 50 value of the HE equals to 13.59 µg/ml HE (containing 12.5 µM total CBD), HE caused OCC specific death, caused OCC specific cytotoxicity, and attenuated OCC proliferation while being safe to NHOECs. HE also decreased OCC migration. Here we concluded that HE has specific anti‐cancer activity against ovarian cancer while being safe to NHOECs. To the best of our knowledge, the ovarian cancer‐cell specific anti‐cancer properties of HE has not been reported to date. The mechanisms behind this specific anti‐cancer properties are currently under investigation.

Marijuana (cannabis sativa), a schedule 1 drug has been recently approved by some of states in the US for its therapeutic benefit. Although few reports discuss about its anti‐cancer potential, currently it has been used mainly for epilepsy and to alleviate pain associated with many diseases. However, due to marijuana's addictive potential there are restrictions for its use. Hemp, which belongs to the same genus and species as marijuana shows similar therapeutic benefits with much less addictive potential due to the low level of the addictive component, tetrahydrocannabinol (THC). Both marijuana and hemp are rich in therapeutically valuable cannabidiol (CBD) and cannabidiolic acid (CBD‐A). The major objective of the current study is to investigate KY hemp's therapeutic potential against ovarian cancer. Our laboratory is interested in searching for alternative therapies for ovarian cancer. As a candidate drug we have investigated the anti‐cancer potential of KY grown hemp using two different epithelial ovarian cancer cell (OCC) lines, which are A2780 and MES‐OV. KY Hemp extracts were made using supercritical CO2 extraction method by KY Commonwealth Extracts Company. Following High Performance Liquid Chromatography (HPLC) analysis by MRX laboratories we have obtained this extract to study its anti‐cancer potential. There are no reports on anti‐cancer activity of KY hemp against ovarian cancer or any type of cancer yet. Therefore, this study provides novelty.MethodsFollowing serum starvation, OCCs were treated with vehicle (negative control), 80 μM doxorubicin and cisplatin, and different concentrations of hemp extracts that contain 8.5–335 μM CBD. Hemp‐induced modulation of ovarian cancer cell viability was assessed spectrophotometrically by MTT cell proliferation assay and Calcein AM assay. The MTT assay detects formazan product formed due to mitochondrial reductase enzyme present in viable cells. The Calcein assay detects the esterase enzyme activity present in viable cells. Ethidium homodimer dye (ETHD‐1) was used to detect total cell death. ETHD‐1 penetrates only damaged cell membranes and binds different molecules such as cell debris, single stranded DNA, double stranded DNA, and peptides. Calcein AM and ETHD‐1 fluorescence was also detected microscopically. KY Hemp extract has significantly decreased OCC proliferation and caused total cell death. KY Hemp extract has shown anti‐cancer properties comparable to doxorubicin and cisplatin. Based on the data here we conclude that KY Hemp has significant anti‐cancer potential against ovarian cancerSupport or Funding InformationFunding is provided by the Sullivan University System via a faculty development grant awarded to Dr. Sumanasekera. Grant number: RG‐1‐PS‐2017‐04