| BackgroundMitochondria is a ubiquitous dynamic organelle in most eukaryotic cells, withdifferent metabolic conditions its shape, size, number and distribution will be different,and it has many characteristics, such as, polymorphism, motility, mutability andadaptability. The primary role of mitochondria is to produce direct energy in the formof adenosine triphosphate (ATP) through the oxidative phosphorylation(OXPHOS).Therefore, mitochondria has " energy factory " laudatory name. when theATP production is insufficient through the OXPHOS process in most malignanttumor cells, it tends to rely more on glycolysis to provide energy to meet the needs ofits rapid growth. But compared with the mitochondria aerobic metabolism, glycolysismetabolism is still very low efficiency in the energy production process. TheOXPHOS process takes place on the electron transport chain (ETC) in the innermitochondrial membrane (IMM). The ETC consists of a series lipoprotein complexeswhich can reversibly receive and release electrons or hydrogen protons, mainlycomprised of four large trans-membrane protein complexes (mitochondrial respiratoryComplex I, II, III and IV). Mitochondria also can generate reactive oxygen species(ROS) as byproducts through the ETC. ROS can generate from complex I, II and III.Cancer cells are frequently under higher levels of intracellular ROS conditions thanthe normal cells. Besides, mitochondria serves as one of the primary sites of apoptosisand autophagy regulation in cancer cells. Autophagy is a programmed cell death mechanism, according to the differentpathways of cellular material transported to the lysosome, can be divided intomacroautophagy, microautophagy and chaperone-mediated autophagy. Generallyspeaking, autophagy means macroautophagy, is a process by whichdouble-membraned autophagosomes parcel intracellular longevity proteins anddamaged organelles, then fuse with lysosomes where they are delivered fordegradation and recycling. Now, autophagy is widely implicated inpathophysiological processes (e.g., cancer, neurodegenerative disorders, andcardiovascular and pulmonary diseases). In recent years, a large number of studieshave been shown that the mitochondrial function is associated with autophagy,including the formation and mature of autophagy. When mitochondrial dysfunction,autophagosomes involved in the degradation of damaged mitochondria, maintainingthe mitochondrial mass, so that the cells can survive.Ursolic acid （UA） are widely present in natural plants and herbs. Ursolic acidhas been found to be low toxicity and have a number of important biological effects,such as anti-cancer, anti-inflammatory, hepato-protective, hypolipidemic, etc.Recently our team identified ursolic acid can induce autophagy-related death in tumorcell, it’s the new anti-tumor mechanisms in ursolic acid. But in this autophagy-relatedcell death, the role of mitochondria is unclear.ObjectiveTo investigate the role of mitochondria in the autophagy-related death inducedby UA on TC-1cancer cells.MethodsIn vitro, TC-1cells were treated with UA (0μM, l0μM,20μM,30μM) for24hrs, then observed the cells number and morphological changes by Opticalmicroscope. The various concentration of UA treatment for1,2,3,4,5days and thendetermined the inhibition of cell proliferation by MTT assay. The mitochondrialmembrane potential, intracellular ATP and ROS level were detected by FCM(flowcytometry）. Observed the activity of the respiratory chain complex enzymes Ⅰ, Ⅱ,Ⅲ andⅣ by spectrophotometry. Mitochondria in TC-1cells were observed by transmission electron microscope(TEM) after UA30μM treatment for6hrs.Fluorescent microscope was used to observe the localization of the mitochondria andLC3, a autophagy marker proteins.ResultsWhen treated for24hrs by low concentration of UA (10μM), TC-1cell show nomorphological changes, when treated by UA (20,30μM), the cells became round andadherent loosely, some little black dots appeared in the cells two poles, and the higherconcentration of UA (20,30μM) induced cell death. UA had a significantantiproliferative effect on TC-1cell in a dose-and time-dependent manner, especiallytreated for3days or more, the inhibition rate of TC-1cell growth more than90%(P＜0．05), detected by MTT. When UA treated for24hours, the negative quadrantfluorescence proportion of total fluorescence were3.1%,3.8%,6.4%,24.7%,respectively. The greater the proportion of negative quadrant, the lower of themitochondrial membrane potential (Δψm), indicated, with the concentrationincreasing, Δψm decreased gradually, especially, UA30μM group, compared withthe control group (P＜0．05). The activity of the complex Ⅰ, Ⅱ, Ⅲ and Ⅳweredecreased observably when UA treated for24hrs, and with the UA concentrationincreasing the activity of the complex decreased gradually, especially complex IV,compared with the control group (P＜0．05). TC-1cells were treated with variousconcentrations of UA for24hrs. We found the ATP level decreased notably, eventhough in UA10μM treatment group, compared with the control group (P＜0．05).In addition, TC-1cells were treated by UA30μM for0,4,8,12,24hrs, respectively,the data showed ATP depleted in a time-dependent manner. After UA treatment for4hrs, the ATP level decreased more than50%compared with the control group. Andthe ATP level closed to0nmol/mg/protein, when treatment for12hrs or more (P＜0．05). The intracellular level of ROS after UA treatment for24hrs shown by thepercentage of positive area of the fluorescence, were about86.46%,82.52%,69.35%,57.24%, respectively (P＜0．05). This means the ROS level decreased graduallywith the UA concentration increasing. Moreover, by using the TEM and fluorescentmicroscope observe whether the mitophagy did exist, it shown when UA (30μM) treated for6hrs, we found mitochondria swelling, cristae fracture and manyfragments of mitochondria. Meanwhile, we found large number of double membranestructures appear in the cytosol, and partly parceled the damaged organelle, especiallyappeared near the damaged mitochondria, which named nascent autophagosomes.Besides, autophagosomes and mitochondria presence co-localization in TC-1cellstreating by UA20μM treated for8hrs.Conclusion1. UA induce TC-1cell death by changing the morphology and function ofmitochondria.2. UA induced Mitophagy in TC-1cell. |
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