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Disporum cantoniense suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis

  • Authors:
    • Chi Feng
    • Yujiao Ji
    • Renqiang Chen
    • Qiuxu Chen
    • Yiru Pan
    • Jianming Wu
    • Dalian Qin
    • Betty Yuen Kwan Law
    • Vincent Kam Wai Wong
    • Xiaogang Zhou
    • Jianxin Liu
    • Anguo Wu
  • View Affiliations / Copyright

    Affiliations: Department of Pharmacy, Suining Chuanshan Hospital of Traditional Chinese Medicine, Sichuan 629000, P.R. China, Sichuan Key Medical Laboratory of New Drug Discovery and Druggability Evaluation, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, Key Laboratory of Medical Electrophysiology of Ministry of Education, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan 646000, P.R. China, Department of Pharmacy, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China, Department of Pharmacy, Ziyang Central Hospital, Ziyang, Sichuan 641300, P.R. China, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau 999078, P.R. China, School of Pharmaceutical Sciences, Sino‑Pakistan Center on Traditional Chinese Medicine, China‑Pakistan International Science and Technology Innovation Cooperation Base for Ethnic Medicine Development in Hunan Province, Hunan University of Medicine, Huaihua, Hunan 418000, P.R. China
    Copyright: © Feng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 137
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    Published online on: October 15, 2026
       https://doi.org/10.3892/ijo.2026.5950
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Abstract

Non‑small cell lung cancer (NSCLC) remains a leading cause of cancer‑associated mortality and urgently requires new therapeutic agents. In the present study, phenotypic screening of natural herbal extracts was performed, identifying Disporum cantoniense extract as a selective inhibitor of NSCLC cell viability. Bioactivity‑guided fractionation further revealed that the petroleum ether fraction (DPF) was the major active fraction. DPF suppressed the viability, proliferation, colony formation, migration and invasion of A549 and H1299 cells while exhibiting weaker cytotoxicity toward BEAS‑2B cells. In addition, DPF increased propidium iodide (PI)‑positive cell death and lactate dehydrogenase release in NSCLC cells, indicating that DPF‑induced viability loss was accompanied by membrane‑damaging cytotoxicity. Transcriptomic and network pharmacology analyses indicated significant enrichment of autophagy‑, apoptosis‑, AMPK‑, mTOR‑, and MAPK‑associated pathways. Mechanistically, DPF increased LC3‑II accumulation, promoted green fluorescent protein‑LC3 puncta formation and autophagic flux, and activated the AMPK/mTOR/unc‑51 like autophagy activating kinase 1 and MEK/ERK signaling pathways. DPF also induced apoptosis, as demonstrated by increased Annexin V positivity, elevated cleaved caspase‑3 and Bax expression, reduced Bcl‑2 expression, and reduced cytotoxicity in Bax‑Bak double‑knockout mouse embryonic fibroblast (MEF) cells. Notably, pharmacological inhibition of autophagy or its upstream regulators attenuated DPF‑induced cytotoxicity. In addition, autophagy‑deficient Atg7‑/‑ MEF cells exhibited reduced DPF‑induced PI‑positive cell death, loss of cell viability, and caspase‑3 activation, further supporting the involvement of Atg7‑dependent autophagy in DPF‑induced apoptosis. In a xenograft model, DPF significantly inhibited tumor growth and increased apoptosis‑ and autophagy‑associated markers in tumor tissues without evident systemic toxicity. Collectively, these findings identify DPF as a principal anti‑NSCLC fraction of Disporum cantoniense and suggest that it suppresses NSCLC progression through AMPK/mTOR/unc‑51 like autophagy activating kinase 1‑ and MEK/ERK‑mediated autophagy‑dependent apoptosis.
View Figures

Figure 1

Cytotoxicity screening in A549 cells
and chemical profiling of Disporum cantoniense. (A and B)
Heatmaps showing cell viability in (A) A549 and (B) BEAS-2B cells
treated with crude herbal extracts. The white arrows indicate DE.
(C and D) Quantification of cell viability in (C) A549 and (D)
BEAS-2B cells treated with different concentrations of DE. (E-H)
Line graphs showing the viability of A549 and BEAS-2B cells treated
with four fractions of DE, including (E) DPF, (F) DEF, (G) DNF and
(H) DWF, at the indicated concentrations. (I) Cell viability of
H1299 cells treated with various concentrations of DPF. (J)
Representative image of Disporum cantoniense and
ultra-high-performance liquid chromatography quadrupole
time-of-flight tandem mass spectrometry chromatograms of DPF
displaying the TIC and the corresponding DAD chromatogram recorded
at 210 nm. Statistical significance is denoted as
**P<0.01 and ***P<0.001. Data are
presented as the mean ± SD of three independent experiments. DE,
Disporum cantoniense extract; DEF, Disporum
cantoniense ethyl acetate fraction; DNF, Disporum
cantoniense n-butanol fraction; DPF, Disporum
cantoniense petroleum ether fraction; DWF, Disporum
cantoniense aqueous fraction; TIC, total ion chromatogram; DAD,
diode-array detector; Ctrl, vehicle; Gefi, gefitinib.

Figure 2

DPF inhibits proliferation, invasion,
colony formation and migration and induces membrane-damaging
cytotoxicity in NSCLC cells. (A and B) Real-time cell analysis
curves showing the cell index of (A) A549 and (B) H1299 cells
treated with DPF at the indicated concentrations over 25 h. (C)
Representative images of Transwell invasion assays in A549 and
H1299 cells following treatment with different concentrations of
DPF (magnification, ×10; scale bar, 200 μm). (D and E)
Quantification of invaded (D) A549 and (E) H1299 cells after DPF
treatment. (F) Representative fluorescence images of Hoechst/PI
staining in A549 and H1299 cells treated with increasing
concentrations of DPF. Nuclei were stained with Hoechst and
PI-positive cells indicate membrane-compromised dead cells (scale
bar, 200 μm). (G and H) Quantification of PI-positive cell
death in (G) A549 and (H) H1299 cells after DPF treatment,
expressed as the percentage of PI-positive cells relative to
Hoechst-stained cells. (I and J) LDH release assay showing
membrane-damaging cytotoxicity in (I) A549 and (J) H1299 cells
following DPF treatment. (K and L) Representative colony formation
images of (K) A549 and (L) H1299 cells treated with DPF at the
indicated concentrations or Gefi. (M) Quantification of the
remaining wound area in A549 cells following DPF exposure. (N and
O) Representative wound-healing images showing the migration of (N)
A549 and (O) H1299 cells at 0 and 24 h after DPF treatment
(magnification, ×10; scale bar, 200 μm). (P) Quantification
of the remaining wound area in H1299 cells. Statistical
significance is denoted as *P<0.05,
**P<0.01 and ***P<0.001. Data are
presented as the mean ± SD of three independent experiments. DPF,
Disporum cantoniense petroleum ether fraction; NSCLC,
non-small cell lung cancer; Gefi, gefitinib; Ctrl, vehicle; LDH,
lactate dehydrogenase; PI, propidium iodide.

Figure 3

Transcriptomic profiling and pathway
enrichment analysis of DPF-treated A549 cells. (A) PCA of
transcriptomic data comparing Ctrl and DPF-treated A549 cells. PC1,
PC2 and PC3 explain 68.34, 18.17 and 5.84% of the variance,
respectively. (B) Volcano plot showing DEGs between Ctrl and
DPF-treated A549 cells. Red points indicate significantly
upregulated genes, while blue points indicate significantly
downregulated genes, with 971 upregulated and 767 downregulated
genes (adjusted P<0.05). (C) Heatmap illustrating the expression
levels of DEGs between Ctrl and DPF-treated A549 cells. Genes are
represented by rows and samples are represented by columns, with
color intensity indicating gene expression levels. (D) GO
enrichment analysis of DEGs comparing Ctrl and DPF-treated A549
cells. The analysis highlights significant biological processes,
including 'apoptotic signaling pathway', 'negative regulation of
cell migration' and 'negative regulation of growth'. (E) KEGG
pathway enrichment analysis of DEGs between Ctrl and DPF-treated
A549 cells. Pathways associated with cell signaling, metabolism and
diseases are significantly enriched in the DPF-treated group. (F)
KEGG pathway enrichment analysis highlighting the most enriched
pathways in DPF-treated A549 cells. Notable pathways include the
'MAPK signaling pathway', the 'mTOR signaling pathway' and pathways
in cancer. DPF, Disporum cantoniense petroleum ether
fraction; PCA, principal component analysis; Ctrl, vehicle; DEGs,
differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto
Encyclopedia of Genes and Genomes; FC, fold change.

Figure 4

(A) Western blotting analysis of
LC3-II/LC3-I ratios in A549 cells treated with DPF at the indicated
concentrations, with Rap serving as a positive control. (B)
Quantification of LC3-II/LC3-I ratios in A549 cells following DPF
treatment. (C) Western blotting analysis of LC3-II/LC3-I ratios in
H1299 cells treated with DPF at the indicated concentrations, with
Rap serving as a positive control. (D) Quantification of
LC3-II/LC3-I ratios in H1299 cells following DPF treatment. (E-H)
Representative fluorescence images of GFP-LC3-expressing (E) A549
and (G) H1299 cells, and tandem monomeric RFP-GFP-LC3
reporter-expressing (F) A549 and (H) H1299 cells treated with DPF
or Rap; nuclei were stained with Hoechst 33342 (magnification, ×63;
scale bars, 20 μm). (I and J) Quantification of GFP-LC3
puncta per cell in (I) A549 and (J) H1299 cells. (K and L)
Quantification of RFP/GFP ratios in (K) A549 and (L) H1299 cells,
indicating changes in autophagic flux. Statistical significance is
denoted as *P<0.05, **P<0.01 and
***P<0.001. Data are presented as the mean ± SD of
three independent experiments. DPF, Disporum cantoniense
petroleum ether fraction; NSCLC, non-small cell lung cancer; Rap,
rapamycin; GFP, green fluorescent protein; RFP, red fluorescent
protein; Ctrl, vehicle.

Figure 5

DPF activates autophagy in NSCLC
cells through AMPK/mTOR/ULK1 and MEK/ERK pathways. (A and B)
Western blotting analysis of phosphorylated and total AMPK, mTOR
and ULK1 proteins in (A) A549 and (B) H1299 cells treated with DPF
at the indicated concentrations, with Rap serving as a positive
control. (C-J) Quantification of (C and G) p-AMPK/AMPK, (D and H)
p-mTOR/mTOR, (E and J) p-ULK1(S555)/ULK1 and (F and I)
p-ULK1(S757)/ULK1 in A549 and H1299 cells. (K and L) Western
blotting analysis of phosphorylated and total MEK and ERK in (K)
A549 and (L) H1299 cells treated with DPF. (M-P) Quantification of
(M and N) p-ERK/ERK and (O and P) p-MEK/MEK in A549 and H1299
cells. (Q) Representative fluorescence images of GFP-LC3 puncta in
A549 cells treated under the following conditions: Ctrl, DPF alone,
CC, ULK101, SCH and their combinations with DPF. Hoechst was used
to stain nuclei (magnification, ×63; scale bar, 20 μm). (R)
Quantification of GFP-LC3 puncta per cell in A549 cells under the
indicated treatments. Statistical significance is denoted as
*P<0.05, **P<0.01 and
***P<0.001. Data are presented as the mean ± SD of
three independent experiments. DPF, Disporum cantoniense
petroleum ether fraction; NSCLC, non-small cell lung cancer; Rap,
rapamycin; GFP, green fluorescent protein; p-, phosphorylated;
Ctrl, vehicle; CC, compound C; SCH, SCH772984; ULK1, unc-51 like
autophagy activating kinase 1.

Figure 6

DPF induces apoptosis in NSCLC cells.
(A) Representative flow cytometry plots showing Annexin V-FITC/PI
staining in A549 and H1299 cells treated with increasing
concentrations of DPF. (B and C) Quantification of total apoptotic
cells in (B) A549 and (C) H1299 cells after DPF treatment. Total
apoptotic cells were calculated as the sum of early and late
apoptotic populations. (D and E) Cell viability of (D) Bax-Bak DKO
and (E) WT MEF cells treated with DPF at the indicated
concentrations. (F and G) Western blotting analysis of Bcl-2, Bax
and caspase-3 in (F) A549 and (G) H1299 cells following DPF
treatment. GAPDH was used as the loading control. (H and I)
Quantification of the Bcl-2/Bax ratio in (H) A549 and (I) H1299
cells. (J and K) Quantification of c-caspase-3/caspase-3 levels in
(J) A549 and (K) H1299 cells. (L) Representative fluorescence
images of caspase-3 activation in A549 cells treated with different
concentrations of DPF. Nuclei were counterstained with Hoechst
(scale bar, 200 μm). (M) Quantification of caspase-3
activation (%) in A549 cells. Statistical significance is denoted
as *P<0.05, **P<0.01 and
***P<0.001. Data are presented as the mean ± SD of
three independent experiments. DPF, Disporum cantoniense
petroleum ether fraction; NSCLC, non-small cell lung cancer; DKO,
double knockout; WT, wild type; MEF, mouse embryonic fibroblast;
PI, propidium iodide; Ctrl, vehicle; c-caspase, cleaved
caspase.

Figure 7

DPF-induced autophagy promotes
apoptotic cell death. (A-D) Cell viability of A549 cells treated
with DPF alone or in combination with (A) the autophagy inhibitor
3-MA, (B) AMPK inhibitor CC, (C) ULK1 inhibitor ULK101 or (D) ERK
inhibitor SCH at the indicated concentrations. (E) Representative
fluorescence images of caspase-3 activation in A549 cells treated
with DPF, 3-MA or their combination. Nuclei were counterstained
with Hoechst (scale bar, 200 μm). (F-I) Quantification of
caspase-3 activation (%) in A549 cells corresponding to panels E
and J. (J) Representative fluorescence images of caspase-3
activation in A549 cells treated with DPF alone or in combination
with CC, ULK101 or SCH (scale bar, 200 μm). (K)
Representative Hoechst/PI staining images of Atg7+/+ and
Atg7−/− MEF cells treated with different concentrations
of DPF (scale bar, 200 μm). (L) Quantification of
PI-positive cell death in Atg7+/+ and Atg7−/−
MEF cells after DPF treatment. (M) Cell viability of
Atg7+/+ and Atg7−/− MEF cells treated with
DPF at the indicated concentrations. (N) Representative
fluorescence images of caspase-3 activation in Atg7+/+
and Atg7−/− MEF cells treated with DPF. Nuclei were
counterstained with Hoechst (scale bar, 200 μm). (O)
Quantification of caspase-3 activation (%) in Atg7+/+
and Atg7−/− MEF cells. Statistical significance is
denoted as *P<0.05, **P<0.01 and
***P<0.001. Data are presented as the means ± SD of
three independent experiments. DPF, Disporum cantoniense
petroleum ether fraction; 3-MA, 3-methyladenine; CC, compound C;
ULK1, unc-51 like autophagy activating kinase 1; SCH, SCH772984;
MEF, mouse embryonic fibroblast; PI, propidium iodide; Ctrl,
vehicle.

Figure 8

DPF suppresses tumor growth in A549
xenograft mice. (A) Representative images of excised tumors from
mice treated with vehicle, Gefi (20 mg/kg) or DPF (40 and 80
mg/kg). (B) Tumor growth curves of A549 xenograft mice in different
treatment groups. (C) Final tumor weights in mice treated with
vehicle, Gefi or DPF at the indicated doses. (D) Body weight
changes of mice during the treatment period. (E) Organ weights of
the heart, liver, spleen, lung and kidney in each group. (F)
Representative H&E and TUNEL staining images of tumor tissues
from mice treated with vehicle, DPF (40 or 80 mg/kg) or Gefi
(magnification, ×10; scale bar, 200 μm). (G) Representative
immunohistochemical staining images of mTOR, ERK, LC3 and Bax in
tumor tissues from each group (magnification, ×10; scale bar, 200
μm). (H-K) Quantitative analysis of (H) mTOR, (I) ERK, (J)
LC3 and (K) Bax expression based on immunohistochemical staining.
Statistical significance is indicated as *P<0.05,
**P<0.01 and ***P<0.001. Data are
presented as the mean ± SD. n=6 mice per group for tumor growth,
tumor weight, body weight and organ weight analyses. DPF,
Disporum cantoniense petroleum ether fraction; Gefi,
gefitinib.

Figure 9

Schematic summary of the anti-NSCLC
effects and underlying mechanism of DPF derived from Disporum
cantoniense. DPF, the active fraction isolated from Disporum
cantoniense, suppresses NSCLC cell proliferation, migration,
invasion and colony formation and induces membrane-damaging
cytotoxicity. Mechanistically, DPF activates AMPK/mTOR/ULK1 and
MEK/ERK signaling to promote pro-death autophagic flux, which
subsequently facilitates Bax/Bak- and caspase-3-dependent
apoptosis. In vivo, DPF inhibits xenograft tumor growth
without evident systemic toxicity. DE, Disporum cantoniense
extract; DEF, Disporum cantoniense ethyl acetate fraction;
DNF, Disporum cantoniense n-butanol fraction; DPF,
Disporum cantoniense petroleum ether fraction; DWF,
Disporum cantoniense aqueous fraction; NSCLC, non-small cell
lung cancer; ULK1, unc-51 like autophagy activating kinase 1;
c-caspase, cleaved caspase.
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Copy and paste a formatted citation
Spandidos Publications style
Feng C, Ji Y, Chen R, Chen Q, Pan Y, Wu J, Qin D, Law BY, Wong VK, Zhou X, Zhou X, et al: <em>Disporum cantoniense</em> suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis. Int J Oncol 69: 137, 2026.
APA
Feng, C., Ji, Y., Chen, R., Chen, Q., Pan, Y., Wu, J. ... Wu, A. (2026). <em>Disporum cantoniense</em> suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis. International Journal of Oncology, 69, 137. https://doi.org/10.3892/ijo.2026.5950
MLA
Feng, C., Ji, Y., Chen, R., Chen, Q., Pan, Y., Wu, J., Qin, D., Law, B. Y., Wong, V. K., Zhou, X., Liu, J., Wu, A."<em>Disporum cantoniense</em> suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis". International Journal of Oncology 69.6 (2026): 137.
Chicago
Feng, C., Ji, Y., Chen, R., Chen, Q., Pan, Y., Wu, J., Qin, D., Law, B. Y., Wong, V. K., Zhou, X., Liu, J., Wu, A."<em>Disporum cantoniense</em> suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis". International Journal of Oncology 69, no. 6 (2026): 137. https://doi.org/10.3892/ijo.2026.5950
Copy and paste a formatted citation
x
Spandidos Publications style
Feng C, Ji Y, Chen R, Chen Q, Pan Y, Wu J, Qin D, Law BY, Wong VK, Zhou X, Zhou X, et al: <em>Disporum cantoniense</em> suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis. Int J Oncol 69: 137, 2026.
APA
Feng, C., Ji, Y., Chen, R., Chen, Q., Pan, Y., Wu, J. ... Wu, A. (2026). <em>Disporum cantoniense</em> suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis. International Journal of Oncology, 69, 137. https://doi.org/10.3892/ijo.2026.5950
MLA
Feng, C., Ji, Y., Chen, R., Chen, Q., Pan, Y., Wu, J., Qin, D., Law, B. Y., Wong, V. K., Zhou, X., Liu, J., Wu, A."<em>Disporum cantoniense</em> suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis". International Journal of Oncology 69.6 (2026): 137.
Chicago
Feng, C., Ji, Y., Chen, R., Chen, Q., Pan, Y., Wu, J., Qin, D., Law, B. Y., Wong, V. K., Zhou, X., Liu, J., Wu, A."<em>Disporum cantoniense</em> suppresses non‑small cell lung cancer through AMPK/mTOR/ULK1‑mediated autophagy‑dependent apoptosis". International Journal of Oncology 69, no. 6 (2026): 137. https://doi.org/10.3892/ijo.2026.5950
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