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. 2021 Apr 6;12(4):365.
doi: 10.1038/s41419-021-03655-2.

AMPK activation by ASP4132 inhibits non-small cell lung cancer cell growth

Ying-Chen Xia #  1 , Jian-Hua Zha #  1 , Yong-Hua Sang #  2 , Hui Yin  1 , Guo-Qiu Xu  1 , Jie Zhen  3 , Yan Zhang  4 , Ben-Tong Yu  5
Affiliations

AMPK activation by ASP4132 inhibits non-small cell lung cancer cell growth

Ying-Chen Xia et al. Cell Death Dis. .

Abstract

Activation of adenosine monophosphate-activated protein kinase (AMPK) is able to produce significant anti-non-small cell lung cancer (NSCLC) cell activity. ASP4132 is an orally active and highly effective AMPK activator. The current study tested its activity against NSCLC cells. In primary NSCLC cells and established cell lines (A549 and NCI-H1944) ASP4132 potently inhibited cell growth, proliferation and cell cycle progression as well as cell migration and invasion. Robust apoptosis activation was detected in ASP4132-treated NSCLC cells. Furthermore, ASP4132 treatment in NSCLC cells induced programmed necrosis, causing mitochondrial p53-cyclophilin D (CyPD)-adenine nucleotide translocase 1 (ANT1) association, mitochondrial depolarization and medium lactate dehydrogenase release. In NSCLC cells ASP4132 activated AMPK signaling, induced AMPKα1-ACC phosphorylation and increased AMPK activity. Furthermore, AMPK downstream events, including mTORC1 inhibition, receptor tyrosine kinases (PDGFRα and EGFR) degradation, Akt inhibition and autophagy induction, were detected in ASP4132-treated NSCLC cells. Importantly, AMPK inactivation by AMPKα1 shRNA, knockout (using CRISPR/Cas9 strategy) or dominant negative mutation (T172A) almost reversed ASP4132-induced anti-NSCLC cell activity. Conversely, a constitutively active AMPKα1 (T172D) mimicked and abolished ASP4132-induced actions in NSCLC cells. In vivo, oral administration of a single dose of ASP4132 largely inhibited NSCLC xenograft growth in SCID mice. AMPK activation, mTORC1 inhibition and EGFR-PDGFRα degradation as well as Akt inhibition and autophagy induction were detected in ASP4132-treated NSCLC xenograft tumor tissues. Together, activation of AMPK by ASP4132 potently inhibits NSCLC cell growth in vitro and in vivo.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. ASP4132 treatment exerts potent anti-NSCLC cell activity.
Primary NSCLC cells that were derived from different patients (pNSCLC-1/-2/-3), the immortalized NSCLC cell lines (A549 and NCI-H1944), as well as primary (“pEpi”) and established (BEAS-2B) lung epithelial cells, were treated with ASP4132 at applied concentrations, cells were further cultured for applied time periods, cell viability (CCK-8 assay, A, G, J), colony formation (B), proliferation (EdU incorporation assay, C, H), cell cycle progression (D), as well as cell migration (E, I) and invasion (F) were tested by assays mentioned in the text, with results quantified. For all EdU assays in this study, five random views containing at least 1, 200 cells of each condition were included to calculate EdU ratio (% vs. DAPI). For all “Transwell” and “Matrigel Transwell” assays in this study, five random views of each condition were included to calculate the average number of migrated/invaded cells. “Veh” stands for the vehicle control (Saline). Data were presented as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “Veh” cells. “n.s.” stands for no statistical difference (J). The experiments were repeated five times with similar results detected. Scale bar = 100 μm (C, E, F).
Fig. 2
Fig. 2. ASP4132 induces apoptosis activation in NSCLC cells.
Primary NSCLC cells that were derived from different patients (pNSCLC-1/-2/-3), the immortalized NSCLC cell lines (A549 and NCI-H1944), as well as primary (“pEpi”) and established (BEAS-2B) lung epithelial cells, were treated with ASP4132 (1 μM) or the vehicle control; Cells were further cultured for applied time periods, caspase activation (A, B, G), single strand DNA (ssDNA) contents (ELISA assays, C) and cell apoptosis (D, E, H, I) were tested by the assays mentioned in the text, with cell death examined by Trypan blue staining assays (F). For all apoptotic nuclei assays in this study, five random views with total 1, 200 cells of each condition were included to calculate the average apoptotic nuclei ratio (% vs. total nuclei). Expression of listed proteins was quantified and normalized to the loading control (B). “Veh” stands for the vehicle control (Saline). Data were presented as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “Veh” cells. “n.s.” stands for no statistical difference (I). The experiments were repeated five times with similar results detected. Scale bar = 100 μm (D, H).
Fig. 3
Fig. 3. ASP4132 activates programmed necrosis cascade in NSCLC cells.
Primary pNSCLC-1 cells were pretreated with the caspase-3 inhibitor z-DEVD-fmk (50 μM, 1 h pretreatment) or the pan caspase inhibitor z-VAD-fmk (50 μM, 1 h pretreatment), followed by ASP4132 (1 μM) treatment and cultured for applied time periods, cell apoptosis (apoptotic nuclei ratio, A), viability (CCK-8 OD, B) and cell death (Trypan blue positive cell ratio, C) were tested. Primary NSCLC cells, pNSCLC-1/-2, were treated with ASP4132 (1 μM) or the vehicle control, cells were further cultured for applied time periods, mitochondrial CyPD-p53-ANT1 association (Mito-IP, D), mitochondrial depolarization (by measuring JC-1 green monomers intensity, E, I) and cell necrosis (by measuring medium LDH contents, F, J) were tested. Expression of CyPD and Tubulin in stable pNSCLC-1 cells with CyPD shRNA lentiviral particles (sh-CyPD) or cyclosporin A (CsA, 5 μM, 24 h) treatment was shown (F). Control cells were treated with vehicle control plus scramble control shRNA lentiviral particles (“shC+DMSO”) (G); Cells were further treated with ASP4132 (1 μM) and cultured for 72 h, cell viability (CCK-8 OD) and cell death (Trypan blue positive cell ratio) were tested (H). Expression of listed proteins was quantified and normalized to the loading control (D, G). “Veh” stands for the vehicle control (Saline). Data were presented as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “Veh” cells. #p < 0.05 vs. “DMSO (0.2%)” pretreatment (A–C). #p < 0.05 vs. “shC + DMSO” cells (H). The experiments were repeated five times with similar results detected. Scale bar = 100 μm (E).
Fig. 4
Fig. 4. ASP4132 activates AMPK signaling in NSCLC cells.
Primary NSCLC cells, pNSCLC-1/-2, were treated with ASP4132 (1 μM) or the vehicle control, cells were further cultured for applied time periods, expression of listed proteins in total cell lysates was tested by Western blotting assays (A, C, D, E); The relative AMPK activity was tested as well (B). List mRNAs were tested by qPCR assays (D). LC3B-II RFP (red fluorescence protein) puncta was examined (F). Expression of listed proteins was quantified and normalized to the loading control (A, C–E). “Veh” stands for the vehicle control (Saline). Data were presented as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “Veh” cells. “n.s.” stands for no statistical difference (D). The experiments were repeated five times with similar results detected. Scale bar = 100 μm (F).
Fig. 5
Fig. 5. AMPK activation mediates ASP4132-induced anti-NSCLC cell activity.
Stable pNSCLC-1 cells, expressing the lentiviral AMPKα1 shRNA (“shAMPKα1”), the CRISPR/Cas9-AMPKα1-KO construct (“koAMPKα1”) (A–D), the dominate negative AMPKα1 (T172A, “dnAMPKα1”) (E–H), the constitutively active AMPKα1 (T172D, caAMPKα1) construct (I–L), or corresponding control shRNA or empty construct, were established; Cells were treated with ASP4132 (1 μM) or vehicle control and cultured for applied time periods, expression of listed proteins was tested by Western blotting assays (A, E, I). Cell viability, apoptosis and death were tested by CCK-8 (B, F, J), apoptotic nuclei staining (C, G, K) and Trypan blue staining (D, H, L) assays, respectively. Expression of listed proteins was quantified and normalized to the loading control (A, E, I). Red stars indicated expression of the mutant AMPKα1 (E, I). “Veh” stands for the vehicle control (Saline). Data were presented as mean ± standard deviation (SD, n = 5). “sh-C + Cas9-C” stands for control pNSCLC-1 cells with scramble control shRNA plus CRISPR/Cas9 empty vector (A–D). “Vec” stands for empty vector (E–L). *p < 0.05 vs. “Veh” cells. #p < 0.05 vs. ASP4132-treatment in “sh-C + Cas9-C” control cells or “Vec” control cells. “n.s.” stands for non-statistical difference. The experiments were repeated five times with similar results detected.
Fig. 6
Fig. 6. Oral administration of ASP4132 inhibits NSCLC xenograft growth in SCID mice.
The SCID mice-bearing pNSCLC-1 xenograft tumors were treated with ASP4132 (oral administration, 5 mg/kg body weight, daily for 21 days) or vehicle control (ten mice per group/n = 10); Estimated tumor volumes (A) and mice body weights (D) were recorded every six days for a total of 42 days; Estimated daily tumor growth was calculated using the described formula (B); At the end of experiments, Day-42, tumors of the two groups were separated via surgery and weighted individually (C). At experimental Day-7 and Day-14, 4 h after ASP4132/vehicle administration, one tumor of each group was isolated and tumor tissues were subjected to Western blotting assay of listed proteins (E–H). Expression of listed proteins was quantified (E–H). Data were presented as mean ± standard deviation (SD).
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