Regulation of Baicalin on Growth of Extranodal NK/T Cell Lymphoma Cells through FOXO3/CCL22 Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of baicalin on the growth of extranodal NK/T cell lymphoma (ENKTCL) cells and its related mechanism.@*METHODS@#Normal NK cells and human ENKTCL cells lines SNK-6 and YTS were cultured, then SNK-6 and YTS cells were treated with 5, 10, 20 μmol/L baicalin and set control. Cell proliferation and apoptosis was detected by Edu method and FCM method, respectively, and expressions of BCL-2, Bax, FOXO3 and CCL22 proteins were detected by Western blot. Interference plasmids were designed and synthesized. FOXO3 siRNA interference plasmids and CCL22 pcDNA overexpression plasmids were transfected with PEI transfection reagent. Furthermore, animal models were established for validation.@*RESULTS@#In control group and 5, 10, 20 μmol/L baicalin group, the proliferation rate of SNK-6 cells was (56.17±2.96)%, (51.92±4.63)%, (36.42±1.58)%, and (14.60±2.81)%, respectively, while that of YTS cells was (58.85±2.98)%, (51.38±1.32)%, (34.75±1.09)%, and (15.45±1.10)%, respectively. In control group and 5, 10, 20 μmol/L baicalin group, the apoptosis rate of SNK-6 cells was (5.93±0.74)%, (11.78±0.34)%, (28.46±0.44)%, and (32.40±0.37)%, respectively, while that of YTS cells was (7.93±0.69)%, (16.29±1.35)%, (33.91±1.56)%, and (36.27±1.06)%, respectively. Compared with control group, the expression of BCL-2 protein both in SNK-6 and YTS cells decreased significantly (P<0.001), and the expression of Bax protein increased in SNK-6 cells only when the concentration of baicalin was 20 μmol/L (P<0.001), while that in YTS cells increased in all three concentrations(5, 10, 20 μmol/L) of baicalin (P<0.001). The expression of FOXO3 protein decreased while CCL22 protein increased in ENKTCL cell lines compared with human NK cells (P<0.001), but the expression of FOXO3 protein increased (P<0.01) and CCL22 protein decreased after baicalin treatment (P<0.001). Animal experiments showed that baicalin treatment could inhibit tumor growth. The expression of CCL22 protein in ENKTCL tissue of nude mice treated with baicalin decreased compared with control group (P<0.01), while the FOXO3 protein increased (P<0.05). In addition, FOXO3 silencing resulted in the decrease of FOXO3 protein expression and increase of CCL22 protein expression (P<0.01, P<0.001).@*CONCLUSION@#Baicalin can inhibit proliferation and promote apoptosis of ENKTCL cell lines SNK-6 and YTS, up-regulate the expression of Bax protein, down-regulate the expression of BCL-2 protein, and down-regulate the expression of CCL22 protein mediated by FOXO3. Animal experiment shown that the baicalin can inhibit tumor growth. Baicalin can inhibit the growth and induce apoptosis of ENKTCL cells through FOXO3/CCL22 signaling pathway.

Dihydromyricetin mediates epithelial mesenchymal transformation and regulates the proliferation and apoptosis of esophageal squamous cell carcinoma cells / 中华肿瘤杂志

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC50) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC50 values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.

Effect of Curcumin on Apoptosis of Acute T-Lymphoblastic Leukemia Cells induced by UMI-77 and Its Related Mechanism / 中国实验血液学杂志

AbstractObjective: To explore the effect and mechanism of curcumin on human T-cell acute lymphoblastic leukemia (T-ALL) cell apoptosis induced by Mcl-1 small molecule inhibitors UMI-77.@*METHODS@#T-ALL cell line Molt-4 was cultured, and the cells were treated with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77 for 24 h. The MTT method was used to detect the cell survival rate after different treatment; According to the results of curcumin and UMI-77, the experimental settings were divided into control group, curcumin group (20 μmol/L curcumin treated cells), UMI-77 group (15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells) and curcumin+ UMI-77 group (20 μmol/L curcumin and 15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells), MTT method was used to detect cell proliferation inhibition rate, Annexin V-FITC/PI double staining method and TUNEL staining were used to detect cell apoptosis, DCFH-DA probe was used to detect cell reactive oxygen species, JC-1 fluorescent probe was used to detect mitochondrial membrane potential, Western blot was used to detect the expression levels of apoptosis-related proteins and Notch1 signaling pathway-related proteins.@*RESULTS@#After the treatment of Molt-4 cells with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77, the cell survival rate was decreased (P<0.05); Compared with the control group, the cell proliferation inhibition rate of the curcumin group and the UMI-77 group were increased, the apoptosis rate of cell was increased, the level of ROS was increased, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, and the protein expression of Bcl-2 was reduced (P<0.05); Compared with the curcumin group or UMI-77 group, the cell proliferation inhibition rate and apoptosis rate of the curcumin+UMI-77 group were further increased, and the level of ROS was increased. At the same time, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, the protein expression of Bcl-2 was reduced (P<0.05); In addition, the mitochondrial membrane potential of the cells after curcumin treatment was decreased, and the proteins expression of Notch1 and HES1 were reduced (P<0.05).@*CONCLUSION@#Curcumin can enhance the apoptosis of T-ALL cells induced by Mcl-1 small molecule inhibitor UMI-77 by reducing the mitochondrial membrane potential, the mechanism may be related to the inhibition of Notch1 signaling pathway.

Effect of Hypoxia Inducible Factor-1α on Chemosensitivity of B-ALL Cells to Vincristine and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia （B-ALL） cells to Vincristine (VCR) and the mechanisms.@*METHODS@#B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl2 pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and β-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and β-actin mRNA levels.@*RESULTS@#CoCl2 could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups.@*CONCLUSION@#Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.

Regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts / 中华烧伤杂志

Objective: To investigate the regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts (HSFbs). Methods: The experimental research methods were used. The 4th-6th passage of HSFbs from human skin were used for the following experiments. HSFbs were co-cultured with sodium ferulate at final mass concentrations of 1, 1×10-1, 1×10-2, 1×10-3, 1×10-4, 1×10-5, and 1×10-6 mg/mL for 48 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and linear regression was used to analyze the half lethal concentration (LC50) of sodium ferulate (n=6). HSFbs were co-cultured with sodium ferulate at final mass concentrations of 0.1, 0.2, 0.3, and 0.4 mg/mL for 24, 48, 72, and 96 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and the cell proliferation inhibition rate was calculated (n=3). According to the random number table, the cells were divided into 0.300 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, 0.003 mg/mL sodium ferulate group treated with sodium ferulate at corresponding final mass concentrations, and negative control group without any treatment. After 72 hours of culture, the cell absorbance values were determined by methyl thiazolyl tetrazolium method (n=5), the microscopic morphology of cells was observed by transmission electron microscope (n=3), the cell apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and the apoptosis index was calculated (n=4), the protein expressions of B lymphocystoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cysteine aspartic acid specific protease-3 (caspase-3) were determined by immunohistochemistry (n=4), and the protein expressions of transformed growth factor β1 (TGF-β1), phosphorylated Smad2/3, phosphorylated Smad4, and phosphorylated Smad7 were detected by Western blotting (n=4). Data were statistically analyzed with one-way analysis of variance and Dunnett test. Results: The LC50 of sodium ferulate was 0.307 5 mg/mL. After being cultured for 24-96 hours, the cell proliferation inhibition rates of cells treated with sodium ferulate at four different mass concentrations tended to increase at first but decrease later, which reached the highest after 72 hours of culture, so 72 hours was chosen as the processing time for the subsequent experiments. After 72 hours of culture, the cell absorbance values in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were 0.57±0.06, 0.53±0.04, 0.45±0.05, respectively, which were significantly lower than 0.69±0.06 in negative control group (P<0.01). After 72 hours of culture, compared with those in negative control group, the cells in the three groups treated with sodium ferulate showed varying degrees of nuclear pyknosis, fracture, or lysis, and chromatin loss. In the cytoplasm, mitochondria were swollen, the rough endoplasmic reticulum was expanded, and local vacuolation gradually appeared. After 72 hours of culture, compared with that in negative control group, the apoptosis indexes of cells were increased significantly in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group (P<0.05 or P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expressions of Bcl-2 of cells in 0.300 mg/mL sodium ferulate group was significantly decreased (P<0.01), the protein expressions of Bax of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.05), and the protein expression of caspase-3 of cells in 0.300 mg/mL sodium ferulate group was significantly increased (P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expression levels of TGF-β1, phosphorylated Smad2/3, and phosphorylated Smad4 of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly decreased (P<0.05 or P<0.01), and the protein expression levels of phosphorylated Smad7 of cells in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.01). Conclusions: Sodium ferulate can inhibit the proliferation of HSFbs of human skin and promote the apoptosis of HSFbs of human skin by blocking the expression of key proteins on the TGF-β/Smad signaling pathway and synergistically activating the mitochon- drial apoptosis pathway.