Miltirone induces cell death in hepatocellular carcinoma cell through GSDME-dependent pyroptosis

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC GSDME-dependent pyroptosis.

Effects of different fixing agents on tumor cell morphology and membrane permeability / 解剖学报

Objective To observe the effect of different fixative solutions on cancer cell morphology and membrane permeability. Methods Human pancreatic acinar epithelial carcinona(HPAC) cells of human pancreatic cancer and HeLa cells of human cervical cancer were fixed with 4 fixation solutions: Freshly prepared 0.25% paraformaldehyde solution; Freshly prepared 4% paraformaldehyde solution; 75% ethanol solution; 90% ethanol solution. The fixation lime is 30 minutes. PBS solution and complete medium were used as the controls. Cell morphology of each group was observed under optical microscope. Changes in cell jncmbrane permeability were observed by fluorescence staining with 7-aminoactinomycin (7-AAD) , which is not cell membrane permeable in intact cells but permeable in damaged cells. Hoechst33342 was used for staining both intact and damaged cells. Results The cells in the complete medium group were similar to unfixed cells in morphology, and the fluorescence staining of 7-AAD was the weakest. The cells in the complete medium group have typical eel! morphology and low 7-AAD permeability. The 0.25% paraformaldehyde solution group had similar cell morphology to the complete medium group, and the 7-AAD fluorescence staining was weak. The morphology of cells in the 4% paraformaldehyde solution group was typical, but the fluorescence staining of 7-AAD was strong. The cells in the 90% ethanol solution group showed swelling, with a larger volume than the unfixed cells and a stronger fluorescence staining of 7-AAD. The cell swelling in 75% ethanol solution group was not as obvious as that in 90% ethanol solution group, and the fluorescence staining of 7-AAD was strong. The cells in PBS group were round, and the fluorescence staining of 7-AAD was strong. Conclusion 0. 25% paraformaldehyde solution can not only fix tumor cells, but also maintain the integrity of cell membrane.

Assessment of Cell Viability in Umbilical Cord Blood by Using 7-AAD/annexin V Dual Staining

BACKGROUND: The quality of cord blood largely depends on cell viability. Viability assessments using trypan blue or 7-aminoactinomycin (7-AAD) staining, which are commonly used methods, may not reflect early apoptosis of cord blood cells. We aimed to investigate early apoptosis in cord blood cells following elapsed time after collection using double staining with annexin V and 7-AAD and to compare the result with that of viability evaluation using trypan blue or 7-AAD staining. METHODS: Umbilical cord blood samples were obtained from 30 pregnant women at the time of delivery between July 2012 and March 2013. Viability of cord blood cells was determined at 0 (T0), 24, and 48 hr after collection by using trypan blue exclusion assay, 7-AAD staining, and 7-AAD/annexin V staining. RESULTS: Viabilities defined by 7-AAD/annexin V staining at T0, 24, and 48 hr after collection were respectively as follows: total nucleated cells, 92.8+/-4.5%, 78.4+/-7.8%, and 65.5+/-8.1%; mononuclear cells, 94.4+/-1.7%, 90.8+/-4.2%, and 84.2+/-6.7%; and CD34-positive cells, 92.4+/-3.0%, 90.7+/-4.7%, and 89.3+/-7.0%. The viability using trypan blue was more than 90% until 48 hr after collection. CONCLUSIONS: The mean viability of total nucleated cells using 7-AAD/annexin V staining decreased to less than 80% at 24 hr after collection; however, the viability of CD34-positive cells was more than 85% until 48 hr. Our study's data will provide useful information for the assessing the quality of cord blood products.

Assessment of Cell Viability in Umbilical Cord Blood before Cryopreservation / 대한수혈학회지

BACKGROUND: The viability of cord blood is an important measure of product quality. Trypan blue (TB) stain is the most commonly and conveniently used method to measure the viability of the cord blood. Recently, cytometric analysis using 7-Aminoactinomycin D (7-AAD) was introduced. Staining with 7-AAD is more sensitive in detecting cellular damage than staining with TB. In addition to this, 7-AAD allows specific measurement of the viability of total nucleated cells (TNC), mononuclear cells (MNC) and CD34+ cells. In this study, we compared the viability of TNC between the TB and 7-AAD method, as well as analyzing the viability of each cell population. METHODS: From February to July 2010, 102 cord blood units were collected and assessed for the viability of TNC by the TB and 7-AAD methods. The viability of mononuclear cells (MNC) and CD34+ cells was assessed by 7-AAD method. RESULTS: The TB and 7-AAD methods were used to assess the viability of TNC, which was 90.1+/-5.7% and 68.4+/-8.0%, respectively. The viability of MNC and CD34+ cells measured by the 7-AAD method was 91.8+/-4.3% and 93.4+/-5.1%, respectively. CONCLUSION: The TNC viability of 7-AAD method was significantly lower than that of TB method. In 7-AAD method, the viabilities of MNC and CD34+ cells were significantly higher than that of TNC. As those are important prognostic factors and measures for successful engraftment after the transplantation, the measurement of the viabilities of MNC and CD34+ cells by 7-AAD method would be helpful to the quality control of the cord blood product.

Effect of G-CSF on Myeloid Leukemic Cell Lines after Treatment with Ara-C / 대한임상병리학회지

BACKGROUND: G-CSF or GM-CSF was frequently used in treatment of acute myeloid leukemia patients. But it has been argued whether this increases the apoptosis of tumor cells in synergy with chemotherapeutic agent, or decreases apoptosis of leukemic cells. We attempted to determine the effect of granulocyte colony-stimulating factor (G-CSF) on leukemic cell line after cytosine arabinoside (Ara-C) treatment. METHODS: KG-1 and HL60 cell lines were incubated with Ara-C for 48 hours, and then washed with media, divided into two groups, one being with the addition of G-CSF and the other being without G-CSF and incubated for another 48 hours. The controls were the same cell lines incubated without Ara-C. The absolute cell counts and apoptotic percentage measured by flowcytometry after stained with 7-aminoactinomycin D (7-AAD) were compared among three groups at 0, 48, and 96 hours. RESULTS: KG-1 cell line showed decreased cell count and increased apoptotic percent in Ara-C/G-CSF and Ara-C group compared with the control group at 48 and 96 hours, and did not showed significant difference between G-CSF group and nonG-CSF group. In HL60, control group showed higher cell count at 48 hours, and G-CSF/Ara-C group showed lower cell count and higher apoptosis than Ara-C group in all trials. CONCLUSIONS: The treatment of G-CSF after Ara-C did not protect apoptosis nor increase the tumor cells in KG-1 and HL60 cell lines. We concluded it would be safe to use G-CSF after administration of chemotherapeutic drug.

Simultaneous Three Color Detection of Surface Antigen (My 7), Intracellular Antigen (c-myc), and DNA Content using Single Laser Flow Cytometry / 대한산부인과학회잡지

Flow cytometry, a useful tool for measuring DNA content and cell differentiation as expressed by cell surface markers, is utilized to measure multiple antigens, especially surface antigen, intracellular oncoprotein, and DNA content, simultaneously. For this simultaneous detection, several methods off ixation and permeabilization have been used with limited values. In this study, 20 ng/ml of lysolecithin in 1% paraformaldehyde solution was utilized for fixation and permeabilization of cultured promyelocytic leukemic cells(HL 60). The cells were first stained with phycoerythrin (PE)-conjugated monoclonal antibody to the cell surface My 7 antigen and then were fixed and permeabilized with 20 ng/ml of lysolecithin in 1% partormaldehyde solution. After incubation, the fixed and permeabilized cells were stained with monoclonal antibody to intracellular c-myc antigen, which were followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The c-myc stained cells were finally stained for DNA content with 7-amino-actinomycin D(7-AAD). This procedure permits excellent staining for intracellular oncoproteins and preservation of surface antigens with relatively low cofficients of variation (CV) for the G0G1 peak of the DNA histograms and suggests that the sequential staining procedure of surface antigen, intracellular antigen, and DNA content will be extended for the study of correlations with cellular differentiation, expression of oncoproteins, and cell cycle analysis in the cells which are obtained from human malignant diseases using a 488 nm single laser flow cytometry.