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Tumor Discovery HHT inhibits pancreatic cancer progress

by incubation at 37°C for 10 min. Unbound CFSE was with 0.1% Tween-20 (TBST) for 1 h at room temperature,
removed by centrifugation. The CFSE-labeled cells were followed by incubation with primary antibodies overnight
then treated with various concentrations of HHT for 24 or at 4°C. The primary antibodies used in this study
48 h. Fluorescence intensity was analyzed using an Accuri included cyclin D2, cyclin-dependent kinase 1 (CDC2),
™
C6 flow cytometer (BD Biosciences, USA). cell division cycle 25c (CDC25c), p53, and GAPDH
(Cell Signaling Technology, USA). After washing with
2.4. Colony-forming assay TBST, the membranes were incubated with horseradish
PANC-1 and Pan02-mCherry cells were seeded into 6-well peroxidase-conjugated secondary antibodies (Biolegend,
plates at a density of 2×10 cells per well. After overnight USA) for 1 h at room temperature. Protein bands were
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adherence, the cells were treated with HHT at different visualized using an enhanced chemiluminescence reagent
concentrations for 48 h, and then the culture medium was (ECL, Merck Millipore, Germany) in a gel imaging system
replaced with fresh medium and replenished every 3 days. (Tanon, China). The blot quantification was performed
After a 14-day incubation, cell colonies were fixed with 4% using ImageJ software (NIH, USA). Protein expression
paraformaldehyde (PFA) and stained with crystal violet levels were normalized to GAPDH, and relative protein
solution. The number of colonies was counted manually. expression levels were calculated.
2.5. In vivo tumor-forming assay 2.8. Cytoskeleton staining assay
Pan02-mCherry cells were cultured with HHT at 25 nM PANC-1 cells were treated with 50 nM HHT for 48 h. After
and 200 nM, respectively. After 24 h, the cells were fixation with 4% PFA, the cells were stained with Flash
™
collected, centrifuged, and counted. Untreated or HHT- Phalloidin Red 594 (Biolegend, USA) for cytoskeleton
treated cells were then subcutaneously injected into mice visualization and DAPI (ZSGB-Bio, China) for nuclear
at a dose of 2×10 /mouse, with four mice per group. staining. Cytoskeletons were observed using an Olympus
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Tumor sizes were recorded during the experiment. On FV1000MPE multiphoton laser scanning microscope
the 23 day post-implantation, in vivo imaging of mouse (Olympus, Japan).
rd
tumors was performed using the Xenogen IVIS Spectrum
system (Caliper Life Sciences, USA). The mice were then 2.9. Intracellular ROS measurement
sacrificed, and the tumors were excised and weighed. After treatment with HHT (50 nM, 100 nM) for various
durations, PANC-1 cells were incubated with 10 μM
2.6. Cell cycle assay 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA,
PANC-1 cells were treated with HHT at 25 nM, 50 nM, and Sigma, USA) for 30 min at 37°C. The fluorescence intensity,
100 nM for 48 h, then collected, washed, and fixed with which represents the intracellular ROS level, was measured
70% ethanol overnight at −20°C. The cells were treated using a flow cytometer. Relative ROS levels were calculated
with RNase for 30 min at 37°C and stained with propidium by normalization to the control.
iodide (PI) staining solution (BD Biosciences, USA) for
30 min. Flow cytometric analysis was performed using an 2.10. Cell OCR measurement
™
Accuri C6 flow cytometer (BD Biosciences, USA), and The OCR of PANC-1 cells was measured using an
the data were processed using ModFit LT software (Verity XFe24 extracellular flux analyzer (Agilent, USA). HHT-
™
Software, USA). pretreated (50 nM, 24 h) and untreated cells were seeded
into XFe24 cell culture microplates and allowed to adhere
2.7. Western blotting overnight. The medium was then replaced with XF base
PANC-1 cells were incubated with HHT at 50 nM and medium supplemented with glucose, pyruvate, and
100 nM for 48 h, then washed with cold PBS and lysed glutamine. OCR measurements were recorded after the
using RIPA buffer (Solarbio, China) supplemented with sequential addition of oligomycin (oligo), carbonyl cyanide
phenylmethylsulfonyl fluoride (PMSF, Solarbio, China) 4-(trifluoromethoxy) phenylhydrazone, and a combination
and protease inhibitors (Applygen, China). The cell of rotenone and antimycin A. The data were analyzed using
lysates were centrifuged at 12,000 rpm for 15 min at 4℃, Seahorse XFe24 software to determine basal respiration,
and the protein concentrations of the supernatants were adenosine triphosphate (ATP)-linked respiration, maximal
determined using a BCA kit (Thermofisher Scientific, respiration, and non-mitochondrial respiration.
USA). Equal amounts of protein were separated on 12%
SDS-PAGE gels and transferred onto 0.45 μm PVDF 2.11. ATP measurement
membranes (Merck Millipore, Germany). The membranes PANC-1 cells were treated with HHT at 50 nM and 100 nM
were blocked with 5% non-fat milk in Tris-buffered saline for 24 or 48 h. ATP levels were measured using an ATP assay

Volume 4 Issue 1 (2025) 101 doi: 10.36922/td.7825