Toxicities Of Oxalicumone A From A Marine-derived Fungus Penicillium Oxalicum In Cultured Renal Epithelial Cells And Human Liver Cells

ObjectiveMalignant tumor is a serious harm to people’s life and health of common diseases, which has become the second cause of death among cardio-cerebrovascular disease. Its incidence increases year by year in recent years, according to the current trend forecast, the number of death caused by malignant tumor will be 8 billion at 2020. At present, the treatment of cancer is not very ideal, and the severity of the hazard and the difficulty of conquering cancer has caused many governments, the scientific community and the public’s great attention. Therefore, to find and develop high-efficiency anti-tumor drugs is still the main way to conquer cancer. Oxalicumone A (POA), a novel dihydrothiophene-condensed chromone, was isolated from marine-derived fungus Penicillium oxalicum. Previous reports implicated that POA showed strong cytotoxicity against human carcinoma, such as:the A375, A549, HeLa, MCF-7, Hep-2, HepG2, SW-620 with the IC50≤10 μM and it had been suggested as a potent anticancer bioactive agent. To research the toxic effect of POA on cultured human normal cell line (L-02), human kidney-2 (HK-2) cells and explore its underlying mechanisms for evaluating its clinical safety, CCK8 assay was used to measure the cell survival rate and Hoechst 33258 staining, flow cytometry, Caspase-3 activity assay and Western-blotting assays were used to observe the changes of cell morphologic, detect cell cycle and apoptosis. DCFH-DA and JC-1 was used to evaluate the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) respectively. We further detect the mechanism of mitochondrial by ATP kit assay and electron microscope observating the internal structure of mitochondria. These studies indicated that POA inhibited L-02, HK-2 cells growth and promoted apoptosis, along with release of ROS, loss of MMP, increased levels of the Cyt-c, Fas and the Bax/Bcl-2 ratio. The results also showed a significant decrease in superoxide dismutase (SOD) activity and glutathione (GSH), nitric oxide synthase (NOS) content accompanied by enhancing the release of N-acetyl-β-D-glucosaminidase (NAG), the leakage of dehydrogenase (LDH), malondialdehyde (MDA) formation, glutamic-pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST) and the release of nitric oxide (NO). All the results revealed that the invitro antiproliferation efficacy of POA on the HK-2 could be observed, through stimulating apoptosis and oxidative stress injury, which may be remarkable to its clinical application.Methods1. To evaluated the inhibition rate of cells induced by POA by using CCK8 kitUsing CCK8 [2-(2-methoxy-4-nitro phenyl)-3-(4-nitro phenyl)-5-(2,4-disulfonic acid benzene)-2 h-tetrazolium monosodium salt] to analyze the cytoxicity of the L-02 and HK-2 cells induced by POA at the indication of yellow formazan product in living cells.2. To observe the change of cell morphology by Hoechst33258Different concentrations of POA dealing with L-02 and HK-2 cells, then Hoechst 33258 dye stained the cells and observe the cell morphological changes under the fluorescence microscope.3. To detect the cell cycle and early apoptosis by PI and AnnexinV/PI stainingUsing flow cytometry instrument technology record cell cycle distribution and apoptosis ratio, Sub-G1 phase and Annexin V+/PI- like cells as a proportion of apoptosis index, study the effect of different doses of POA on L-02 and HK-2 cell apoptosis.4. To detect the influence of Caspase family activityUsing the Caspase-3 activity detection kits to detect the Caspase-3 activity in different groups cells induced by different concentrations of POA and evaluate of the effect of POA to Caspase family activity in the L-02 and HK-2 cells.5. To detect the AST, ALT, LDH, NAG, SOD, MDA, GSH, NO, NOSAfter POA exposed to the L-02 and HK-2 cells for 24 h, taking culture supernatant or cell lysis solution, according to the method described in the kit of SOD, MDA, LDH, GSH, NOS, NO, AST, ALT were determined, detecting the effects of oxidation pressure balance induced by POA on L-02 and HK-2 cells.6. To detect the reactive oxygen species (ROS)Reactive oxygen species (ROS) including oxygen free radicals, hydrogen peroxide and its downstream products of peroxide and hydroxide, etc., involved in cell growth, proliferation, differentiation of development, aging and apoptosis, as well as many physiological and pathological process. Using active oxygen scavenger DCFH-DA and flow cytometry instrument technology to detect the changes of cell active oxygen value induced by POA.7. To detect the expression of apoptosis proteins by Western-blotingBy using western blot method, through the specific antibody and gel electrophoresis analyzing the the shader of Bax, Bcl-2 and Fas protein,which represent the quantities of the protein expression, to reflect the proteins associated with mitochondrial pathway in the L-02 and HK-2 cells induced by POA.8. To detect the Mitochondrial membrane potential (MMP), ATP, mitochondrial membrane permeable holes (MPTP) value, Cyt-C protein expressionCollect cells samples described by reagent method detection of fluorescence intensity, using fluorescent dye JC-1, calculating the influence of MMP on L-02 and HK-2 cells induced by POA; Using the ATP, MPTP detection kits, according to the different methods described in the kits about sample testing, at the same time we use Western-bloting technology to detect the expression of Cyt-C protein related to mitochondrial, summarizing the influence of POA on the mitochondrial pathway of L-02 and HK-2 cells.Results1. The inhibition rate of L-02 and HK-2 cell induced by POAThe POA showed a typical concentration- and time-dependent manner compared with the control. And DMSO did not show any inhibitory effect on the cell viability. About the L-02 cells, in the 24 h period, the inhibition present significant increasing trend in the range of 0 μM-60 μM; in the 48 h and 72 h periods, it represented obvious dose correlation in the range of 0 μM-60 μM; when the dose reached 60 μM,the inhibition in plateau stage; when the concentration of POA from 60 μM to 100 μM, the inhibition close to the maximum. About the HK-2 cells, its effective inhibition concentration is 20 μM-100 μM; in the study, the three time periods are represented obvious dose correlation, while the inhibitory effect on the HK-2 cells into the plateau stage when the dose concentration reached to 90 μM and close to the maximum. In the results of the studies, it showed that at the 24 h,48 h and 72 h three time periods POA represented inhibition on L-02 and HK-2 cells and the effect showed significant difference (P< 0.05) after statistics data.2. The morphological changes of cells induced by POAWith the POA (0,20 and 40 μM) exposed to L-02 and HK-2 cells:we observed that the normal L-02 looks like epithelioid cells and the normal HK-2 cells in paving stone look, and the cells are clear boundary, live to connect and adherent; while the death cell morphology with cell shrinkage, the boundary is not clear, and appear some cracking phenomenon under inverted phase contrast microscope. Under the fluorescence microscope, normal living cells in a weak blue, cell distribution more uniform, and the boundary is clear; while the apoptotic cells showed pyknotic nuclei rupture, light blue, and thick color increased with the increasing of the concentration of POA.3. The cell cycle and early apoptosis of cells induced by POAAbout the L-02 cells, the POA (10,20 and 40 μM) groups compared to the control group, the proportions of Sub-Gl, S and G2/M phase cells increased significantly (P< 0.01), while the cell ratio of Gl phase was significantly decrease (P< 0.001), which showed obviously that POA caused the L-02 cells apoptosis. With Annexin V-FITC for the horizontal axis, PI for the longitudinal axis, the early apoptosis rate (Annexin V+/PI-) were 10.5%,16.4%,20.1%. Obviously, with the increase of content of POA the cell early apoptosis rate increased gradually, which in a concentration-dependent manner.About the HK-2 cells, the POA (20,40 and 80 μM) groups compared to the control group, the proportions of Sub-G1, S and G2/M phase cells increased significantly (P< 0.01), while the cell ratio of G1 phase was significantly decrease (P< 0.001), which showed obviously that POA caused the L-02 cells apoptosis. With Annexin V-FITC for the horizontal axis, PI for the longitudinal axis, the early apoptosis rate (Annexin V+/PI-) were 3.4%,23.0%,30.8%. Obviously, with the increase of content of POA the cell early apoptosis rate increased gradually, which in a concentration-dependent manner.4. The activity of Caspase-3 of cells induced by POAAbout the L-02 cells, the Caspase-3 activity of POA (10,20 and 40 μM) groups is 1.32,1.44,1.62 folds of the control group respectively. Apparently, the activity of Caspase-3 rised with the POA concentration increased, and dose were positively correlated (P< 0.001), which suggested that POA cause L-02 cell apoptosis in the death process.About the HK-2 cells, the Caspase 3 activity of POA (20,40 and 80μM) groups is 1.19、1.43、1.71 folds of the control group respectively. Apparently, the activity of Caspase-3 rised with the POA concentration increased, and dose were positively correlated (P< 0.001), which suggested that POA cause HK-2 cell apoptosis in the death process.5. The influence of oxidative stress balance induced by POAThe study about the L-02 and HK-2 cells induced by POA, the result showed that compared with the blank control group, POA reduced the activities of antioxidant enzymes GSH, SOD and the content of NO and NOS, while increased the quantities of MDA, LDH, NAG, ALT and AST, which suggested that POA destroyed the cell oxidative stress balance.6. The reactive oxygen species (ROS) induced by POAAbout the L-02 cells, the ROS production value of POA (10,20 and 40 μM) is 14.2, 19.5,19.5 respectively, and the blank control group has a value of 4.2. Apparently, the ROS production amount rises as the rise of the drug concentration, which showed that the POA damage L-02 cells and caused the increase of ROS production.About the HK-2 cells, the ROS production value of POA (20,40 and 80 μM) is 3.8, 6.2,15.2 respectively, and the blank control group has a value of 0.9. Apparently, the ROS production amount rises as the rise of the drug concentration, which showed that the POA damage L-02 cells and caused the increase of ROS production.7. The influence of the expression of apoptosis proteinsAfter the POA exposed to L-02 and HK-2 cells, compared with control group, POA significantly increased the expression of Fas and Bax apoptosis protein (P< 0.01), while reduced the expression of anti-apoptosis protein Bcl-2 (P< 0.001), and it in a concentration-dependent manner, which suggested that the apoptosis induced by POA involved in mitochondria signaling pathways.8. The mechanism of mitochondrial damage induced by POAAbout the L-02 cells, exposed to POA (10,20 and 40 μM) for 24 h, the decline percentage of MMP is 15.1%,20.3% and 26.1%, respectively, and the value of the blank control group is 10.4%; while about the HK-2 cells, exposed to POA (20,40 and 80 μM) for 24 h, the decline percentage of MMP is 14.8%,16.2% and 26.5%, respectively, and the value of the blank control group is 12.3%. The value of ATP declined as the rise of the drug concentration and the MPTP increase with the rising of the drug concentration; transmission electron microscope observed that cavity internal structure in cell mitochondria and the area of the cavity increased as the increase of the drug concentration. Compared with the control group, the Cyt-C content in cytoplasm rised as the drug concentration increases. In a word, POA induced damage to L-02 and HK-2 cell mitochondria and it will be serious as the rise of drug concentration.ConclusionThe POA which derived from marine organisms has cytotoxic effect to normal liver and kidney cells and in a topical time- and concentration-dependent manner. The mechanism of cytotoxicity may be related to the regulation of apoptosis proteins and through the mitochondrial apoptotic pathways. What is more, the mechanism related to the unbalance of the oxidative stress, the other mechanisms about the cytotoxic effect remains to be further studied.