EFFECTS OF CARDANOL FROM Apis mellifera PROPOLIS ON BT-474 BREAST CANCER CELL LINE

Abstract

Propolis is one of products from honeybee. It is sticky resin like plant resin and has many bioactivities. Thus, in research, cardanol, an anti-proliferative compound against breast cancer BT-474 cells was isolated from Apis mellifera propolis in Nan province, Thailand. Crude propolis was extracted by three organic solvents which were methanol, dichloromethane, and hexane. Later, it was purified by quick column and adsorption chromatography. The obtained cardanol was proved by thin layer chromatography and mass spectrometry. Due to 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay and the calculated inhibition concentration at 50% (IC50), the expected cardanol could be really active against BT-474 cells with the IC50 value of 15.57 ± 1.73 µg/ml. Cardanol led to the morphology change of treated BT-474 cells such as cell shrinkage, cell detachment from substratum which, later, caused the cell death. In order to investigate the program cell death, cells were stained with annexin V and propidium iodide (PI). It was found that cells were dead via late apoptosis, including both apoptosis and necrosis. Next, cells were stained by PI in order to determine cell cycle arrest. It was shown that cell cycle was arrested at G1 subphase. Moreover, the change in expression of genes involving in cell division was observed at both transcriptional and translational levels in order to know a mechanism on how cardanol could inhibit the proliferation. For the former, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used. For the latter, western blot was used. It was found that cardanol induced the arrest in cell cycle at G1 subphase. The obtained data from qRT-PCR and western blot was coincided to the data of qRT-PCR in term that cardanol increased the expression level of ERK, JNK, and p38. Also, it up-regulated the p21 expression. In contrast, it could decrease the expression of cyclin D1 and CDK4 at G1 subphase. Also, it decreased the expression of cyclin E and CDK2 which were important for the cell cycle to continue to the S subphase. Hence, within cells, no DNA replication and cell division occurred. That led to the death of BT-474 cells.