Betulinic Acid Mediates Apoptosis In Saccharamyces Cerevisiae And The Association Study Of Vacuolar Protease Pep4p

Betulinic acid, a type of pentacyclic triterpene acid, has a wide range of plant sources. It has a variety of biological activities, such as anti-tumor, anti-HIV, anti-infl ammatory, antimicrobial, and antimalarial activity. People also find that betulinic acid possesses other special biological activities like anti-high cholesterol and anti-obesity. Recent years, it was highly regarded for its anti-tumor activity. Moreover, the remarkable selectivity cytotoxicity induced by BetA in a diversity of cancer types in conjunction with its minimal cytotoxicity for normal cells has raised this compound as an optimistic candidate for a non-toxic drug lead compound. However signal path of mammalian cells was intricate with a lot of interference factors. The investigation of the exact mechanisms underlying the remarkable anti-cancer potential of BetA is still a challenge for researchers, and the direct molecular target(s) of BetA remain largely to be clarified. Apoptosis is an organized suicide program which is evolutionally conserved from yeast to mammals, and the advantages of yeast model for apoptosis research are reviewed. Yeast has been validated as a model organism to investigate mechanisms of apoptosis instead of complex mammalian cells. Meanwhile this would offer assistance for clarify the regulatory mechanism of betulinic acid inducing mammalian cells-apoptosis.Pep4p in vacuole was the orthologue of the human CatD which could catalyze proteins hydrolysis and play a role in transcription of other enzymatic. Recently researchers found that vacuolar Pep4p participated in the regulation of yeast cell apoptosis and interfered with mitochondrial degradation together with mitochondrial AAC proteins. Thus, we discussed the interplay between vacuolar protease Pep4p and mitochondria in yeast-PCD. It will be exceedingly useful in the elucidation of some yet unclarified roles of lysosomes and CatD in mammalian apoptosis.This paper mainly focused on the following four contents,1. The toxic effect of betulinic acid on yeast cells;2. Whether betulinic acid induced yeast death in the mode of apoptosis;3. The mechanism of betulinic acid inducing yeast apoptosis;4. The role of Pep4p in the process of betulinic acid inducing yeast apoptosis.In this study,20μg/mL betulinic acid was used to impact on yeast cells. Cell viability was tested by Cell Blue Cell Viability Assay Kit. Plant experiment was used to determine the cell viability of S. cerevisiae. Then DAPI staining, Annexin V/PI staining and TUNEL staining were used to detect apoptosis of yeast cells. The intracellular organelles structures of yeast were observed by transmission electron microscope (TEM). The results showed that with treating time increasing, cell viability declined and the mortality rate of yeast increased, accompanied with hallmarks of apoptosis like generation of nuclear fragment, chromatin contraction, phosphatidylserine ectropion and DNA breakage. It indicated that the mortality of yeast cell was induced by betulinic acid through apoptosis. Dihydroethidium (DHE) and rhodamine123were used to detect the generation of ROS and mitochondrial transmembrane potential, respectively; Florescent real-time quantitative (q-PCR) was used to evaluate the expression changes of six apoptosis genes, they are Aifl, Ndil, Cyc1, Cyc7, Fis1and Dnml. The results showed that the generation of ROS was increased and mitochondrial membrane potential hyperpolarized followed by a depolarization. The gene expression levels in yeast cells were obvious changed after treatment with betulinic acid. Betulinic acid could significantly inhibit the viability of yeast cells, and induce apoptosis via mitochondrial pathway. What’s more, the existence of Pep4p could protect the cells from betuliniic acid, retarding the harmful effect of betulinic acid to yeast cells.