Journal: Cancer Cell International

Article Title: Loss of PR55α promotes proliferation and metastasis by activating MAPK/AKT signaling in hepatocellular carcinoma

doi: 10.1186/s12935-021-01796-0

Figure Lengend Snippet: Knockdown of PR55α by shRNA activates AKT and ERK1/2 signaling. a Microarray analysis was conducted to screen the mRNAs which were subjected to PR55α regulation. b KEGG pathway analysis revealed the potential signaling pathways involved in PR55α. c AKT/ERK signaling pathway protein expressions in HCC cells transfected with shPR55α or shControl were evaluated using western blot. d The indicated proteins of AKT/ERK signaling pathway were detected with or without AKT inhibitor LY294002 (20 μM) and MEK inhibitor U0126 (15 μM)

Article Snippet: Five HCC cell lines and one normal liver cell line L02 were purchased from KeyGen (Nanjing KeyGen Biotech Co, Ltd, Jiangsu, China).

Techniques: Knockdown, shRNA, Microarray, Protein-Protein interactions, Transfection, Western Blot

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 treatment induced rapid cIAPs degradation but failed to induce cell death in HepG2 and HCCLM3 cells. (A,B) The mRNA expression of cancer stem cell (CSC) genes ( ABCG2, CD44 , and SOX2 ) and the percentages of side population (SP) cells in HepG2 and HCCLM3 cells, were measured by qRT-PCR and flow cytometry, respectively. (B) The proportion of SP cells in HepG2 and HCCLM3 cell lines were 0.16% and 32.2%, which reduced to 0% and 0.28% in the presence of verapamil. (C) The relative expression of mRNA of CSC and IAP genes between SP and the main population (MP) HCCLM3 cells were examined by qRT-PCR. (D) The basic protein levels of cIAP1, cIAP2, and XIAP in HepG2 and HCCLM3 cells were analyzed by Western blot. (E,F) The changes in protein levels of the IAPs and cleaved poly (ADP-ribose) polymerase (PARP) proteins, (G,H) and cell viability of HepG2 and HCCLM3 cells were detected after treatment with AGP-1387 as a single agent at indicated concentrations and time periods. Con, control; cl-PARP, cleaved-PARP; kDa, kilodalton. Error bars represented the mean ± SEM of triplicate representative experiments; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, by two-tailed unpaired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 treatment enhanced TNF-α and TRAIL mediated anti-cancer activities in the HCC cell lines. (A,B) HepG2 and (C,D) HCCLM3 cells were pre-inoculated in quadruplicate at 2,000 cells per well in 96-well plates for 12 h, cell viability was evaluated using a CCK-8 assay after 24 h of stimulation with TNF-α or TRAIL, or in combination with APG-1387. (E) HepG2 and (F) HCCLM3 cells were pre-seeded in 6-well plates at 1,000 cells per well for 12 h and then stimulated with 2 μM APG-1387, 100 ng/ml TNF-α, 20 ng/ml TRAIL or their combination. After incubating for 14 days, the colonies were stained with Giemsa dye and counted with ImageJ software. (G,H) After 24 h of stimulation with APG-1387 and TNF-α or TRAIL, the percentages of side population (SP) cells were analyzed by flow cytometry after staining with Hoechst 33342 fluorescence dye alone or in combination with verapamil, which was used as a control to block the efflux of Hoechst 33342. (I) Under indicated treatment of APG-1387 and TNF-α or TRAIL, lysates of HepG2 and HCCLM3 cells were harvested, then the level of Sox2 protein was evaluated using a Western blot assay. Con, control; A, 2 μM APG-1387; T, 100 ng/ml TNF-α; TR, 20 ng/ml TRAIL; kDa, kilodalton. Error bars represented the mean ± SEM of triplicate representative experiments; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, by two-tailed unpaired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: CCK-8 Assay, Staining, Software, Flow Cytometry, Fluorescence, Control, Blocking Assay, Western Blot, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 treatment enhanced TNF-α- and TRAIL-induced cell death in HepG2 and HCCLM3 cells. (A–C) HepG2 and HCCLM3 cells were seeded at 1 × 10 6 cells per well in 6-well plates for 12 h. After 24 h of stimulation with APG-1387 and TNF-α or TRAIL, all of the cells were collected separately, and incubated with Annexin-V and 7-AAD for 15 min, then apoptosis (Annexin-V+ 7-AAD–) and necrosis (Annexin-V+ 7-AAD+) of HepG2 or HCCLM3 cells were detected by flow cytometry. (D) After 24 h of stimulation with APG-1387 and TNF-α or TRAIL, lysates of HepG2 and HCCLM3 cells were harvested. Western blot was performed to analyze the changes in levels of apoptosis-related proteins, including poly (ADP-ribose) polymerase (PARP), caspase-3, cleaved-caspase-8, cleaved-caspase-9, cIAP1, cIAP2, and XIAP and gasdermin E (GSDME). (E,F) HepG2 and HCCLM3 cells were pre-treated with a pan-caspase inhibitor (20 μM Z-VAD-fmk) or RIPK1 inhibitor (50 μM Nec-1) for 1 h. Then combination treatments involving APG-1387 with TNF-α or TRAIL were used, and the percentages of cell death were evaluated by flow cytometry. Con, control; A, 2 μM APG-1387; T, 100 ng/ml TNF-α; TR, 20 ng/ml TRAIL; cl-PARP, cleaved-PARP; Cas - 3, caspase-3; cl-Cas - 3, cleaved-caspase-3; cl-Cas - 8, cleaved-caspase-8; cl-Cas - 9, cleaved-caspase-9; GSDME-F, full-length GSDME; GSDME-N, N terminal fragment of GSDME; Nec-1, necrostatin-1; kDa, kilodalton. Error bars represented the mean ± SEM of triplicate representative experiments; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, by two-tailed unpaired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: Incubation, Flow Cytometry, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 sensitized HepG2 and HCCLM3 cells to NK cell-mediated killing in vitro. HepG2 or HCCLM3 cells were co-cultured with purified NK cells, which were stimulated with 10 ng/ml interleukin (IL)-12, 10 ng/ml IL-15, and 100 ng/ml IL-18, either alone or in the presence of 2 μM APG-1387. (A,B) After co-incubation for 24 h, HepG2 or HCCLM3 cells were collected for analysis of killing effect of NK cells by flow cytometry, (C) while supernatants were collected for the LDH assay. Con, control; A, 2 μM APG-1387; NK(+), NK cells co-cultured with 10 ng/ml IL-12, 10 ng/ml IL-15, and 100 ng/ml IL-18. Error bars represented the mean ± SEM of triplicate representative experiments; ∗ p < 0.05, ∗∗ p < 0.01; by paired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: In Vitro, Cell Culture, Purification, Incubation, Flow Cytometry, Lactate Dehydrogenase Assay, Control

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 sensitized HCCLM3 tumors toward NK cell-mediated killing in vivo. Four groups of NOD-SCID mice bearing human HCC cell line HCCLM3 tumors were injected intraperitoneally with APG-1387 at 20 mg/kg every 3 days for 4 weeks, and/or injected at the tumor site with 2 × 10 7 IL-12, IL-15, and IL-18-activated NK cells per mouse (or with the same volume of PBS) every 6 days for 4 weeks. (B,E) Tumor volume and body weight were measured every 3 days. (C,D) After 4 weeks of treatment, the mice were sacrificed, the tumors were harvested and weighed. (A) The expression of IAPs in tumors was measured by Western blot and (F) the relative proportion of cleaved caspase-3 was shown using confocal microscopy. Con, control; A, APG-1387; NK(+), NK cells co-cultured with 10 ng/ml IL-12, 10 ng/ml IL-15, and 100 ng/ml IL-18; cl-Cas-3, cleaved-caspase-3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, N.S., not significant; by two-tailed unpaired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: In Vivo, Injection, Expressing, Western Blot, Confocal Microscopy, Control, Cell Culture, Two Tailed Test