Journal: Cancer Biology & Medicine

Article Title: VDAC1 protein derived from extracellular vesicles promotes paclitaxel resistance in gastric cancer through autophagy and mitophagy

doi: 10.20892/j.issn.2095-3941.2025.0360

Figure Lengend Snippet: EV-derived VDAC1 protein activates autophagy by modulating AMPK/mTOR/p70S6K signaling to promote the PTX resistance of GC cells. (A) Intracellular ATP levels were measured following VDAC1 overexpression in PTX-sensitive cells (AGS-OE) and VDAC1 knockdown in PTX-resistant cells (AGSR). (B) GSEA analysis of VDAC1 knockdown in PTX-resistant cells (AGSR). (C) Western blot analysis of p-AMPK, p-p70S6K, p-mTOR, and p-ULK1 in AGS and AGSR cells. (D) Western blot analysis of p-AMPK, p-p70S6K, p-ULK1, and p-mTOR in AGSR cells with or without VDAC1 knockdown. (E) Western blot analysis of p-AMPK, p-p70S6K, p-ULK1, and p-mTOR in AGSR cells with or without VDAC1 knockdown in the presence of Rapa (100 nM). (F) Western blot analysis of p-AMPK, p-ULK1, p-mTOR, and p-p70S6K in AGS cells after pre-incubation with EVs-shNC or EVs-shVDAC1. (G) TEM analysis of autophagosomes in AGS cells after pre-incubation with EVs-shNC or EVs-shVDAC1. AMPK, Adenosine 5′-monophosphate (AMP)-activated protein kinase; EV, extracellular vesicle; mTOR, mammalian target of Rapamycin; p70S6K, p70 ribosomal protein S6 kinase; PTX, paclitaxel; Rapa, Rapamycin; TEM, transmission electron microscopy; ULK1, unc-51 like autophagy activating kinase 1; VDAC1, Voltage-dependent anion channel protein 1.

Article Snippet: The primary antibodies used in western blots were as follows: β-actin (1:1000, T002; Affinity, Cincinnati, OH, USA); GAPDH (1:5000, 104941-AP; Proteintech, Wuhan, China); P-gp (1:3000, 22336-1-AP; Proteintech); CD9 (1:1000, sc-13118; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HSP90 (1:5000, 13171-1-AP; Proteintech); calnexin (1:1000; c-23954; Santa Cruz Biotechnology, Inc. USA); TSG101 (1:1000, c-7964; Santa Cruz Biotechnology, Inc.); VDAC1 (1:1000, PTM-6157; PTM Biolabs, Chicago, IL, USA); p62 (1:1000, #5114; Cell Signaling Technology, Danvers, Massachusetts, USA); LC3B (1:1000, L7543; Sigma); ULK1 (1:1000, 20986-1-AP; Proteintech); p-ULK1 (1:1000, #14202; Cell Signaling Technology); AMPK (1:1000, #5831; Cell Signaling Technology); p-AMPK (1:1000, #2535; Cell Signaling Technology); mTOR (1:5000, 20657-1-AP; Proteintech); p-mTOR (1:2000, 67778-1-Ig; Proteintech); p70S6K (1:2000, 14485-1-AP; Proteintech); p-p70S6K (1:1000, #AF3228; Affinity); ATG5 (1:1000, 10181-2-AP; Proteintech); ATG7 (1:1000, 10088-2-AP; Proteintech); HTRA1 (1:1000, 55011-1-AP, Proteintech); and STEAP4 (1:1000, 11944-1-AP; Proteintech).

Techniques: Derivative Assay, Over Expression, Knockdown, Western Blot, Incubation, Transmission Assay, Electron Microscopy

Journal: Cancer Biology & Medicine

Article Title: VDAC1 protein derived from extracellular vesicles promotes paclitaxel resistance in gastric cancer through autophagy and mitophagy

doi: 10.20892/j.issn.2095-3941.2025.0360

Figure Lengend Snippet: Clinical significance of VDAC1 in GC. (A) Representative IHC images of VDAC1 and P-gp expression in PTX-sensitive and -resistant patients ( n = 34). (B) Statistical comparison of VDAC1 and P-gp expression between 17 PTX-sensitive and 17 PTX-resistant patients. (C) Correlation analysis of VDAC1 and P-gp expression in 17 PTX-sensitive vs. 17 PTX-resistant patients. (D) Representative IHC images of LC3B, p-AMPK, p-mTOR, p-p70S6K, and p-ULK1 expression in PTX-sensitive and -resistant patients ( n = 34). (E) Statistical comparison of LC3B, p-AMPK, p-mTOR, p-p70S6K, and p-ULK1 expression between 17 PTX-sensitive and 17 PTX-resistant patients. (F) Correlation analysis of LC3B, p-AMPK, p-mTOR, p-p70S6K, p-ULK1, and VDAC1 expression in 17 PTX-sensitive vs. 17 PTX-resistant patients. AMPK, Adenosine 5‘-monophosphate (AMP)-activated protein kinase; EV, extracellular vesicle; GC, gastric cancer; IHC, Immunohistochemistry; mTOR, mammalian target of Rapamycin; P-gp, P-glycoprotein; p70S6K, p70 ribosomal protein S6 kinase; PTX, paclitaxel; ULK1, unc-51 like autophagy activating kinase 1; VDAC1, voltage-dependent anion channel protein 1.

Article Snippet: The primary antibodies used in western blots were as follows: β-actin (1:1000, T002; Affinity, Cincinnati, OH, USA); GAPDH (1:5000, 104941-AP; Proteintech, Wuhan, China); P-gp (1:3000, 22336-1-AP; Proteintech); CD9 (1:1000, sc-13118; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HSP90 (1:5000, 13171-1-AP; Proteintech); calnexin (1:1000; c-23954; Santa Cruz Biotechnology, Inc. USA); TSG101 (1:1000, c-7964; Santa Cruz Biotechnology, Inc.); VDAC1 (1:1000, PTM-6157; PTM Biolabs, Chicago, IL, USA); p62 (1:1000, #5114; Cell Signaling Technology, Danvers, Massachusetts, USA); LC3B (1:1000, L7543; Sigma); ULK1 (1:1000, 20986-1-AP; Proteintech); p-ULK1 (1:1000, #14202; Cell Signaling Technology); AMPK (1:1000, #5831; Cell Signaling Technology); p-AMPK (1:1000, #2535; Cell Signaling Technology); mTOR (1:5000, 20657-1-AP; Proteintech); p-mTOR (1:2000, 67778-1-Ig; Proteintech); p70S6K (1:2000, 14485-1-AP; Proteintech); p-p70S6K (1:1000, #AF3228; Affinity); ATG5 (1:1000, 10181-2-AP; Proteintech); ATG7 (1:1000, 10088-2-AP; Proteintech); HTRA1 (1:1000, 55011-1-AP, Proteintech); and STEAP4 (1:1000, 11944-1-AP; Proteintech).

Techniques: Expressing, Comparison, Immunohistochemistry

Journal: Cancer Biology & Medicine

Article Title: VDAC1 protein derived from extracellular vesicles promotes paclitaxel resistance in gastric cancer through autophagy and mitophagy

doi: 10.20892/j.issn.2095-3941.2025.0360

Figure Lengend Snippet: Proposed model for the role of EV-derived VDAC1 in mediating PTX resistance in GC. (Left) VDAC1 is packaged into extracellular vesicles (EVs) in PTX-resistant GC cells. These EVs are internalized by PTX-sensitive cells, where VDAC1 activates the AMPK signaling pathway. Activated AMPK inhibits mTOR, leading to ULK1 activation, which subsequently induces autophagy and mitophagy. This process confers PTX resistance to recipient cells. (Right) The VDAC1 inhibitor, DIDS, blocks the function of EV-derived VDAC1 protein in recipient cells. This process prevents AMPK activation, maintains mTOR activity, and suppresses ULK1. Consequently, inhibition of autophagy and mitophagy restores cellular sensitivity to PTX. This finding highlights the therapeutic potential of inhibiting EV-mediated VDAC1 transfer. AMPK, adenosine 5′-monophosphate (AMP)-activated protein kinase; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; EV, extracellular vesicle; GC, gastric cancer; mTOR, mammalian target of rapamycin; PTX, paclitaxel; ULK1, Unc-51-like autophagy activating kinase 1; VDAC1, voltage-dependent anion channel protein 1.

Article Snippet: The primary antibodies used in western blots were as follows: β-actin (1:1000, T002; Affinity, Cincinnati, OH, USA); GAPDH (1:5000, 104941-AP; Proteintech, Wuhan, China); P-gp (1:3000, 22336-1-AP; Proteintech); CD9 (1:1000, sc-13118; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HSP90 (1:5000, 13171-1-AP; Proteintech); calnexin (1:1000; c-23954; Santa Cruz Biotechnology, Inc. USA); TSG101 (1:1000, c-7964; Santa Cruz Biotechnology, Inc.); VDAC1 (1:1000, PTM-6157; PTM Biolabs, Chicago, IL, USA); p62 (1:1000, #5114; Cell Signaling Technology, Danvers, Massachusetts, USA); LC3B (1:1000, L7543; Sigma); ULK1 (1:1000, 20986-1-AP; Proteintech); p-ULK1 (1:1000, #14202; Cell Signaling Technology); AMPK (1:1000, #5831; Cell Signaling Technology); p-AMPK (1:1000, #2535; Cell Signaling Technology); mTOR (1:5000, 20657-1-AP; Proteintech); p-mTOR (1:2000, 67778-1-Ig; Proteintech); p70S6K (1:2000, 14485-1-AP; Proteintech); p-p70S6K (1:1000, #AF3228; Affinity); ATG5 (1:1000, 10181-2-AP; Proteintech); ATG7 (1:1000, 10088-2-AP; Proteintech); HTRA1 (1:1000, 55011-1-AP, Proteintech); and STEAP4 (1:1000, 11944-1-AP; Proteintech).

Techniques: Derivative Assay, Activation Assay, Activity Assay, Inhibition

Journal: Aging (Albany NY)

Article Title: Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease

doi: 10.18632/aging.101759

Figure Lengend Snippet: Geniposide treatment decreases mTOR activation markers in brains of APP/PS1 mice. Hippocampal expression of Akt, mTOR, and 4E-BP1, and their respective phosphorylated forms was detected by western blot. The expression of p-Akt ( A ) and p-mTOR ( B ) was enhanced in APP/PS1 mice compared to WT, and geniposide attenuated this increase. The expression of p-4E-BP1 ( C ) in APP/PS1 mice was reduced compared to WT, and geniposide partly restored this decrease. Data are presented as mean ± SEM (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05 vs. WT; # p < 0.05 vs. APP/PS1 mice (one-way ANOVA, Tukey's Multiple Comparison Test). WT: wild-type mice. GP: geniposide.

Article Snippet: The membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.05% Tween-20) for 1h, and incubated overnight at 4°C with primary antibodies directed against: Akt (1:1,000), p-Akt (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), 4E-BP1 (1:1,000), or p-4E-BP1 (1:2,000), followed by incubation at 4°C for 2h with a goat-anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Comparison