ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Viscum articulatum Burm. f. (leafless mistletoe) has been used in traditional system of medicines in India, China, Taiwan, Cambodia, Laos, and Vietnam, to treat blood-related diseases and various inflammatory and degenerative diseases including cancer. Anticancer activities of some phytomolecules purified from Viscum articulatum Burm. f. have been tested. However scientific evidence for the anticancerous potential of aqueous extract of V. articularum (VAQE) used in traditional medicine is lacking. AIM OF THE STUDY: To study the antiproliferative and apoptotic effect of VAQE on Jurkat E6.1 and THP1 leukemia cells. MATERIALS AND METHODS: The aqueous extract of the whole plant of Viscum articulatum Burm. f. was prepared in phosphate buffer saline. In VAQE, total soluble protein was estimated using Bradford's dye-binding assay; flavonoid content was determined using aluminum chloride colorimetric assay; and phenolic content was estimated following Folin-Ciocalteu colorimetric assay. XTT cell viability assay was used to test VAQE induced cytotoxicity in Jurkat E6.1 and THP1 leukemia cells and peripheral blood mononuclear cells (PBMC). The effect of VAQE on cell cycle progression was analyzed by PI staining using flow cytometry. Annexin-V-FITC/PI differential staining method was used for detecting the onset of apoptosis in leukemia cells. Rhodamine 123 dye was used to detect the change in mitochondrial membrane potential (MMP) using flow cytometry. DCF-DA fluorescence dye was used to estimate the level of reactive oxygen species (ROS). The ROS inhibitors were used to evaluate the role of ROS in mediating DNA degradation in VAQE-treated leukemia cells. The molecular mechanisms underlying VAQE induced apoptosis induction was studied by analyzing the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins, caspase-8 and caspase-3 enzymes using western blot. Diphenylamine (DPA) assay was used to determine the DNA fragmentation and conclusion of apoptosis. RESULTS: VAQE triggered cytotoxic effect on Jurkat E6.1 (IC50-2.4⯵g/ml; 24â¯h) and THP1 (IC50-1.0⯵g/ml; 24â¯h) cells in a dose- and time-dependent manner. The apoptosis induction and G2/M arrest of the cell cycle are the cause of VAQE-induced cytotoxicity in leukemia cells. The apoptosis in VAQE-treated Jurkat E6.1 and THP1 cells was mediated via a reduction in MMP, elevation of intracellular ROS, decreased expression of the anti-apoptotic (Bcl-2) and increased expression of the pro-apoptotic (Bax) protein, activation of caspase-8 and caspase-3 and DNA fragmentation. CONCLUSION: VAQE has a high efficacy to exert a cytotoxic effect in Jurkat E6.1 and THP1 cells and to induce apoptosis and G2/M cell cycle arrest. VAQE induces extrinsic pathway of apoptosis in both the leukemia cell lines via disruption of MMP, intracellular ROS imbalance, increased ratio of Bax/Bcl-2, activation of caspase-8, caspase-3 and ROS-mediated DNA fragmentation. The knowledge gained from the outcomes of the study may encourage the identification of novel chemotherapeutic agent from Viscum articulatum Burm. f. to treat leukemia.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Plant Extracts/pharmacology , Viscum , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/physiology , Cell Cycle Checkpoints/physiology , Cell Proliferation/physiology , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Humans , Jurkat Cells , Plant Extracts/isolation & purification , THP-1 CellsABSTRACT
Leukemia is among the most aggressive and prevalent human malignant carcinoma. Chemotherapy is the preferred therapeutic strategy; however, recurrence of cancer and non-selective cytotoxicity are the major concerns. Unlike synthetic chemotherapeutic agents, mistletoe ribosome-inactivating protein (RIP) displays anti-tumor function in various types of cancers. However, its effect on leukemia cells is little explored. In this study, we assessed the impact of Viscum articulatum RIP (Articulatin-D) on the survival of acute T-cell leukemia cells and the involved molecular and cellular mechanisms. Cell proliferation assay showed that Articulatin-D suppressed the viability of leukemia cells selectively. We further confirmed that the elevation of mitochondrial membrane potential and exposure of phosphatidylserine are the early events of apoptosis induction in Articulatin-D-treated Jurkat cells. Subsequently, we found that Articulatin-D treatment induces apoptosis in Jurkat cells in a time- and concentration-dependent manner. In conclusion, we provided evidence that Articulatin-D efficiently activates caspase-8 involved in extrinsic pathway of apoptosis induction, which ultimately results in caspase-3-dependent DNA fragmentation of Jurkat cells. Further evaluation of Articulatin-D in cell culture and animal models may provide novel information on selective cytotoxicity to acute T-cell leukemia and its involvement in targeting tumor cell survival pathways.