| Hepatocellular carcinoma (HCC) is one of the most common and malignant tumors. Currently, chemotherapy is a main way for cancer treatment. However, the efficacy is limited by the deficient types of drugs, the drug resistance as well as the imperfect mechanisms. As long as arsenic trioxide (As2O3) been successfully used for the treatment of acute promyelocytic leukemia, great interest has aroused around the role of As2O3 in clinical studies in solid tumors. Recent studies have shown that As2O3 can inhibit HCC through reduction of angiogenesis, inhibition of cell proliferation, reduction of cell invasion and metastasis as well as induction of apoptosis and autophagy. Among all the pathways, proliferation inhibition is the basic pathway of As2O3-induced hepatoma cell toxicity. Mitochondrion, as an important and ubiquitous intracellular organelle, impacts well in the regulation of oncocyte proliferation due to the changes of its structure, function and quantity. Meanwhile, mitochondrion is also a primary target in As2O3-stimulated toxic response. However, whether cell proliferation could be suppressed through mitochondrion pathway under As2O3 exposure and the possible mechanisms are poorly understood.In the present study, the human hepatoma cell line, HepG2 were chosen to explore the effects of As2O3 on cell proliferation and mitophagy. Two mitophagic inhibitors, cyclosporin A (CsA) and mitochondrial division inhibitor-1 (Mdivi-1), as well as the specific cyclooxygenase-2 (COX-2) inhibitor, NS398 were used to further illustrate the underlying mechanisms on inhibition of proliferation via mitophagy pathway. Some techniques were used for the research, such as MTT assay, colony formation assay, real-time quantitative PCR analysis, transmission electron microscope observation, immunoblotting, immunocytochemistry. The present research would supply more toxicity data for clinical use of As2O3 as an anticancer drug.The results showed that:1. As2O3 (2,4,8 μM) suppressed the viability of HepG2 cells in a dose-and time-dependent manner during different treatment times (12,24,36 h) (P< 0.05), showing that the exposure conditions of As2O3 used in the present study caused toxic effect in HepG2 cells.2. After cells were exposed to different treatment doses or times of As2O3, cell growth, cell colony-forming efficiency, as well as cell proliferation activity (manifested by Ki-67 mRNA level) reduced in a dose-and time-dependent manner (P< 0.05). However, no apoptosis was observed in any group (P> 0.05). The above researches showed that As2O3 inhibited HepG2 cell proliferation in a dose- and time-dependent manner, which is not related to cell apoptosis.3. Using transmission electron microscope, mitophagosomes and mitolysosomes were observed in cells exposed to 4 μM As2O3 at 12 and 24 h. Besides, a dose- and time-dependent increase of both the protein level of the full-length PINK1 and the LC3 Ⅱ/Ⅰ ratio were detected (P< 0.05). Moreover, the number of visible colocalization which contains mitochondrion and lysosome increased, whereas, COX Ⅳ expression level reduced in a dose- and time-dependent manner (P< 0.05). The above results showed that As2O3 induced mitophagy in HepG2 cells in a dose- and time-dependent manner, meanwhile the mitochondrial mass was reduced as well.4. Pretreatment of cells with CsA (5 μM) or Mdivi-1 (5 μM) for 2 h significantly reduced PINK1 level and LC3 Ⅱ/Ⅰ ratio, while increased COX Ⅳ and Ki-67 mRNA levels (P< 0.05). The above results showed that the cell proliferation of HepG2 was inhibited through mitophagy under As2O3 exposure.5. The protein and mRNA levels of COX-2 were suppressed by As2O3. In cells treated with COX-2 inhibitor, NS398 (100 μM), cell proliferation was extremely significantly inhibited, as manifested by Ki-67 mRNA level (P< 0.01). These results recovered that COX-2 did play a positive function on the inhibition of cell proliferation under As2O3 exposure.6. The significant recovery of COX-2 protein levels was observed in cells pretreated with CsA/Mdivi-1, compared with that in cells treated with As2O3 alone (P< 0.05), which showed that mitophagy could reduce the accumulation of COX-2 protein in HepG2 cells. Besides, the subcellular localization of COX-2 showed that a majority of COX-2 located in mitochondria whereas only a trace amount was detected in cytoplasm. Treatment with As2O3 reduced COX-2 in both mitochondrial and cytosolic fractions. Pretreatment with CsA increased COX-2 protein levels in both parts and were even higher than that in control group. On the contrary, pretreatment with Mdivi-1 reduced COX-2 protein levels in mitochondrial fractions, while the levels in cytosolic fractions changed little. However, the mRNA levels of COX-2 increased significantly both in cells pretreated with CsA and Mdivi-1, compared with that in As2O3-treated group (P< 0.01). The above results showed that along with the mitochondrial autophagic degradation, a mass mount of COX-2 localized in mitochondria were degraded as well, meanwhile, mitophagy could further inhibit the transcription and translation of COX-2.In conclusion, the present investigations indicated that As2O3 could induce mitophagy in HepG2 cells, which further reduced COX-2 protein levels and hence inhibited cell proliferation. During which, along with the elimination of mitochondria by mitophagy, the COX-2 which were localized in mitochondria were degraded as well. Specifically, in some extent, mitophagy would inhibit the expression of COX-2. |
PDF Full Text Request