The AMPK inhibitor compound C is a potent AMPK-independent antiglioma agent

Abstract

AMP-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor important for cell growth, proliferation, survival, and metabolic regulation. Active AMPK inhibits biosynthetic enzymes like mTOR and acetyl CoA carboxylase (required for protein and lipid synthesis, respectively) to ensure that cells maintain essential nutrients and energy during metabolic crisis. Despite our knowledge about this incredibly important kinase, no specific chemical inhibitors are available to examine its function. However, one small molecule known as compound C (also called dorsomorphin) has been widely used in cell-based, biochemical, and in vivo assays as a selective AMPK inhibitor. In nearly all these reports including a recent study in glioma, the biochemical and cellular effects of compound C have been attributed to its inhibitory action toward AMPK. While examining the status of AMPK activation in human gliomas, we observed that glioblastomas express copious amount of active AMPK. Compound C effectively reduced glioma viability in vitro both by inhibiting proliferation and inducing cell death. As expected, compound C inhibited AMPK; however, all the antiproliferative effects of this compound were AMPK independent. Instead, compound C killed glioma cells by multiple mechanisms, including activation of the calpain/cathepsin pathway, inhibition of AKT, mTORC1/C2, cell-cycle block at G2-M, and induction of necroptosis and autophagy. Importantly, normal astrocytes were significantly less susceptible to compound C. In summary, compound C is an extremely potent antiglioma agent but we suggest that caution should be taken in interpreting results when this compound is used as an AMPK inhibitor.

Figure 1. Compound C is a potent anti-glioma agent

(A) Chemical structure of Compound C. (B) Immunoblots showing pACC levels in glioma cells treated with DMSO (control) or Compound C. (C) Histogram showing the dose-dependent effect of Compound C (1, 2.5, 5 and 10μM) on the viability of three glioma cell lines. Numbers inside bars represent % dead cells. (D) Histogram showing the dose-dependent effect of Compound C (5 and 10μM) on the viability of three patient-derived primary GBM sphere cultures. (E) Proliferation assay showing the anti-proliferative effect of Compound C on glioma cells relative to normal astrocytes. * P ≤ 0.001. Data shown is representative of three to five independent experiments.

Figure 2. The antiproliferative effect of Compound C is AMPK-independent

Immunoblots showing the expression of the AMPK β1 and β2 subunits in T98G glioma cells (A) and the effects of silencing the β1 subunit in reducing AMPK activity (B). Actin was used as a loading control. (C) Proliferation assay showing the effects of Compound C (5 and 10 μM) in control (nt) and β1 shRNA expressing T98G glioma cells. * P ≤ 0.001. Data shown is representative of three independent experiments.

Figure 3. Compound C is a pleiotropic agent that affects multiple cellular pathways

(A) Immunoblots showing the effect Compound C (5 and 10 μM) on phosphorylation of mTOR effectors (S6 and 4EBP1). In (B), eIF4E was immunoprecipitated with m7GDP-sepharose and bound 4EBP1 was detected with 4EBP1 antibody. (C-E) Immunoblots showing the effects of Compound C (5 and 10μM) in glioma cells on Akt phosphorylation (C), apoptosis (D) and autophagy (E); Note, increased processing of LC3A/BI to LC3A/BII in (E). DMSO was used as control. (F) Flow cytometry-based apoptosis/necrosis analysis of glioma cells treated with Compound C (5μM and 10μM). DMSO was used as control. * P ≤ 0.005. Data is representative of two to three independent experiments.

Figure 4. The cellular effects of Compound C are AMPK-independent

(A) Immunoblot showing the effect Compound C (10μM) on phosphorylation of mTOR effectors (S6 and 4EBP1) in control (nt) and AMPK β1-silenced glioma cells. In (B), eIF4E was immunoprecipitated with m7GDP-sepharose and bound 4EBP1 was detected with 4EBP1 antibody in control (nt) and AMPK β1-silenced glioma cells. (C-E) Immunoblots showing the effects of Compound C on Akt phosphorylation (C), apoptosis (D) and autophagy (E) in control (nt) and AMPK β1-silenced glioma cells. (F) Flow cytometry-based apoptosis/necrosis analysis of control (nt) and AMPK β1-silenced glioma cells treated with Compound C (5μM and 10μM). DMSO was used as control. * P ≤ 0.005. Data is representative of two to three independent experiments.

Figure 5. Compound C inhibits glioma cell migration independent of AMPK

(A) Digital photographs of T98G glioma cells treated with DMSO (control) or Compound C (5 and 10μM), and (B) that of nt (control) and AMPK β1-silenced glioma T98G cells treated with Compound C (10μM) for indicated times. Note that control cells completely filled in the gap in 48 hours which was potently inhibited by Compound C in (A) and this inhibition was essentially similar in nt and β1 shRNA cells in (B). Data is representative of three independent experiments.

Figure 6. Compound C blocks glioma cell cycle at G2M independent of AMPK

(A, B) Histograms and (C) quantitation of flow cytometry-based cell cycle analysis showing glioma cells in G2M phase that were treated for 24 hours with either DMSO (control) or Compound C (10μM). Histograms showing G2M stage occupancy of control (nt) (D, E) and AMPK β1-silenced (F, G) T98G cells treated with DMSO (control) or Compound C. (H) Quantitation of data shown in (D-G). * P < 0.001. Data shown is representative of two independent experiments.

Figure 7. A common Calpain/Cathepsin inhibitor partially rescues glioma cells from Compound C's inhibitory action

Viability assay of T98G glioma cells treated with DMSO (control) or Compound C (5μM) in the presence of indicated doses of ALLN, a compound that inhibits Calpain I, Calpain II, cathepsin B and cathepsin L. * P < 0.001. Data shown is representative of two independent experiments.