Cannabinoid: A Potential Anti-Cancer Agent for Non-Hodgkin Lymphoma
Type of DegreePhD Dissertation
General Veterinary Medicine
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The non-Hodgkin lymphomas (NHLs) are a heterogeneous family of lymphoid malignancies and are one of the most commonly diagnosed neoplasm in both dogs and humans. Systemic anticancer chemotherapy is the treatment of choice for human and canine lymphomas. Canine and human lymphoma are generally characterized by a high rate of initial remission following conventional CHOP (cyclophosphamide, hydroxyl-doxorubicin, vincristine, and prednisone) based therapies; however, 95% of dogs and 30% of humans will succumb to drug-resistant relapse and in most cases face severe side effects of chemotherapeutic drugs. Owing to shared molecular, signaling, incidence, and pathologic features, treatment approach and treatment need, studying novel targets for lymphoma treatment in canine can be beneficial for both species. Cannabis or Marijuana have been used as medicine for centuries. With renewed interest in the cannabinoid (CBs) as a medicine, in the last two decades, cannabinoids have been extensively studied for their anti-cancer effects in various models of cancers including NHL, and have demonstrated promising effects against tumor growth, angiogenesis and metastasis. However, the anti-cancer effects of cannabinoids have never been studied in canine lymphoma and there is very limited literature available on the canine endocannabinoid system. Finding this wide-open area to study in canine lymphoma our aim of this research was to 1. Study the expression of cannabinoid receptors CB1 and CB2, 2. Analyze and compare the anti-cancer effects of cannabinoids in human and canine NHL cell lines and 3. To study the effect of cannabinoids in combination with traditional NLC drugs and compare it with the effect of CBs and NLC drugs alone. To study the expression of cannabinoid receptors CB1 and CB2, canine B cell type (1771 and CLBL1) and T cell type (CL1) NHL cell lines, canine PMBCs and, human B cell type NHL cell line (Ramos) were used. Cells were cultured in RPMI, and receptor expression was studied using real-time PCR. Our results demonstrated positive expression of cannabinoid receptor CB1 and/or CB2 in both canine and human lymphoma cell lines, with a significantly higher expression of CB1 and CB2 receptors in canine and human B cell lymphoma cell lines, compared to activated PBMCs and canine CL-1 lymphoma cell line. For activated canine PBMCs our results show negative expression of the CB1 receptor gene but significantly higher expression of CB2 receptor gene compared to the canine T cell lymphoma cell line. After establishing the expression of cannabinoid receptors, anti-cancer effects of cannabinoids [Endocannabinoids (AEA, 2AG), phytocannabinoids (CBD, THC) and synthetic cannabinoid (WIN)] on canine and human NHL cell lines were analyzed using MTT cell viability assay. Cells were treated at concentrations from 0.1µM to 50µM for 24 and 48 hours. Results of the cell viability assay demonstrate a dose-dependent decrease in cancer cell viability as compared to the control (cells treated with vehicle only) with AEA and CBD in both B and T cell type NHL cell lines. Treatment with WIN and THC showed dose-dependent decrease in cell viability in only B-cell NHL cell lines. 2-AG appeared to decrease cell viability in only lymphoma cell lines with higher CB2 receptor expression (Ramos and CLBL-1). To further confirmed the anti-cancer effects of cannabinoids we selected canine B cell lymphoma cell line 1771 and exposed it to 0 (vehicle only) and 1µM and 50µM concentrations of cannabinoids for 24 h and protein was extracted. Markers of oxidative stress, mitochondrial function and apoptosis analyzed using biochemical spectrophotometric and fluorometric analysis. Results of the biochemical analysis revealed a cannabinoid-induced increase in markers of oxidative stress and apoptosis and a decrease in markers of mitochondrial functions in cells treated with cannabinoids (AEA, CBD, THC and WIN) as compared to the control. To study the effect of cannabinoids in combination with traditional NHL chemotherapeutic drugs (NLC drugs), canine 1771 lymphoma cells were treated with CBs (AEA, CBD and WIN) and NLC drugs (DOX, CYC, VIN, LOM and PRD) alone and combinations. The cytotoxicity of each drug alone and combinations was analyzed by MTT assay and combination effect was analyzed using combinational index (CI) analysis. Our results demonstrated that the cytotoxic effects of all traditional NHL chemotherapy drugs were synergistically enhanced (interaction with CI <1) by each of the three cannabinoids when added to 1771 canine malignant B type NHL cells. Taken together, these studies show the anti-cancer potential of cannabinoids against both canine and human NHL and provides a resource for developing therapeutics and testing safety prior to initiating canine and human studies.