1.Study on regulation of ethanol extract of Turkish Galls on JAK 2/STAT3 signaling pathway in colorectal cancer cells
Elham ALIYA ; Arken ABDULAZIZ ; Bo YAN ; Anwar ARFIDIN ; Iminjan MUBARAK
China Pharmacy 2022;33(3):326-331
OBJECTIVE To study the regulatory mechanism of ethanol extract of Turkish Galls on proliferation and migration of colorectal cancer cells HCT- 116 and Caco- 2 based on janus kinase 2(JAK2)/signal transducer and activator of transcription 3 (STAT3)signaling pathway. METHODS CCK-8 method was used to detect the effects of 0.05,0.1,0.2,0.3,0.4 and 0.5 mg/mL ethanol extract of Turkish Galls on the proliferation of HCT- 116 and Caco- 2 cells after treated for 12,24,48 and 72 h. After treated with 0.1,0.3,0.5 mg/mL ethanol extract of Turkish Galls for 24 h,the migrations of HCT- 116 and Caco- 2 cells were detected by scratch test ;the level of reactive oxygen species (ROS)was detected by fluorescent probe method. The levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in cell supernatant were detected by ELISA . The phosphorylations of JAK2 and STAT 3 as well as the expressions of B-cell lymphoma- 2(Bcl-2)and Bcl- 2 associated protein X (Bax)were detected by Western blot assay. RESULTS Compared with blank control ,0.05,0.1,0.2,0.3,0.4 and 0.5 mg/mL ethanol extract of Turkish Galls could significantly inhibit cell proliferation after treated for 12,24,48,72 h(P<0.05). After treated with 0.1,0.3 and 0.5 mg/mL ethanol extract of Turkish Galls for 24 h,the scratch healing rate of 2 kinds of cells ,the levels of IL- 6 and TNF-α in the cell supernatant ,the phosphorylation of JAK 2 and STAT 3 as well as the expression of Bcl- 2 protein were all significantly decreased (P<0.05);the level of ROS and protein expression of Bax were increased significantly (P<0.05). CONCLUSIONS The ethanol extract of Turkish Galls can inhibit the proliferation and migration of HCT- 116 and Caco- 2 cells. The mechanism may be related with down-regulation of protein expression of Bcl- 2 and up-regulation of protein expression of Bax by increasing the accumulation of intracellular ROS ,down-regulating the expressions of inflammatory factors IL- 6 and TNF-α and the phosphorylation of JAK2 and STAT 3 in JAK 2/STAT3 signaling pathway.
2.Study on Anti-colorectal Cancer Mechanism of Gallic Acid Based on JAK/STAT Pathway
Elham ALIYA ; Arken ABDULAZIZ ; Kalimanjan GULINA ; Anwar ARFIDIN ; Iminjan MUBARAK
Chinese Journal of Experimental Traditional Medical Formulae 2022;28(4):91-99
ObjectiveTo investigate the effects of gallic acid (GA) on human colon cancer HCT-116 and Caco-2 cell activities, intracellular Janus kinase (JAK)/signal transducer and activator of transcription factor (STAT) signaling pathway, and the expression of anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and pro-apoptotic protein B-cell lymphoma-2-associated X protein (Bax), so as to explore its underlying molecular mechanism. MethodFollowing the classification of cells into GA group, blank group, and 5-fluorouracil (5-FU, 0.05 g·L-1) group, the HCT-116 and Caco-2 cells were treated with GA (0.02, 0.05, 0.1, 0.15, 0.2 g·L-1) for 12, 24, 48, and 72 h, respectively, and the cell proliferation inhibition rats were determined by cell counting kit-8 (CCK-8) assay to select the GA concentration that effectively inhibited proliferation. The colony formation ability was detected by crystal violet staining and the migration of cells by scratch test. The level of reactive oxygen species (ROS) was measured using a fluorescent probe (DCFH-DA). The expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell supernatant were determined using the enzyme-linked immunosorbent assay (ELISA) kits. The expression levels of JAK2, phosphorylated (p)-JAK2, STAT3, p-STAT3, Bcl-2, and Bax were assayed by Western blot. ResultCCK-8 assay showed that after 12, 24, 48, and 72 h of treatment, GA (0.02, 0.05, 0.1, 0.15, 0.2 g·L-1) inhibited the proliferation of HCT-116 and Caco-2 cells in a dose- and time-dependent manner, and the inhibition rates were higher than those in the blank control group. Compared with the 5-FU group, GA (0.2 g·L-1) enhanced the inhibition of cell proliferation in a time-dependent manner. Compared with the blank control group, GA (0.1, 0.15, and 0.2 g·L-1) significantly decreased the number of cell colonies (P<0.01), increased the inhibition rate of cell colony formation (P<0.01), diminished the scratch healing rate (P<0.05, P<0.01), elevated the fluorescence intensity of intracellular ROS (P<0.01), and down-regulated the expression of IL-6 and TNF-α in the supernatant (P<0.01) in a dose-dependent manner. Compared with the 5-FU group, GA (0.2 g·L-1) decreased the scratch healing rate (P<0.01), enhanced the fluorescence intensity of intracellular ROS (P<0.01), and down-regulated the levels of IL-6 and TNF-α in cell supernatant (P<0.01). According to Western blot analysis, compared with the blank control group, GA (0.1, 0.15, 0.2 g·L-1) obviously lowered the expression of p-JAK2, p-STAT3, Bcl-2, p-JAK2/JAK2, p-STAT3/STAT3, and Bcl-2/Bax (P<0.01) and raised Bax protein expression (P<0.05, P<0.01) in a dose-dependent manner. Compared with the 5-FU group, GA (0.2 g·L-1) down-regulated the expression of p-JAK2, p-STAT3, Bcl-2, p-JAK2/JAK2, p-STAT3/STAT3, and Bcl-2/Bax (P<0.05, P<0.01) and up-regulated the expression of Bax protein (P<0.05, P<0.01). ConclusionGA significantly inhibits the proliferation of HCT-116 and Caco-2 cells, which may be related to the increased accumulation of intracellular ROS, down-regulation of inflammatory factors IL-6 and TNF-α, p-JAK2 and p-STAT3 protein expression in JAK/STAT signaling pathway, and Bcl-2, and up-regulation of Bax.