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Tumor Discovery HHT inhibits pancreatic cancer progress

ranks 12 in new cases but sixth in cancer mortality 2. Materials and methods
th
worldwide. Among pancreatic cancers, pancreatic ductal
2
adenocarcinoma (PDAC) has one of the highest mortality 2.1. Cells, reagents, and animal models
rates among solid tumors, with an overall 5-year survival Human pancreatic cancer cell lines PANC-1 and SW1990,
rate of <10%. Pancreatic tumors treated with standard mouse pancreatic cancer cell line Pan02-mCherry, and
3
chemotherapy regimens are prone to developing drug mouse macrophage cell line RAW264.7 were purchased
resistance, whose mechanisms are further complicated from the Cell Resource Center, Institute of Basic Medical
4
by the genetic heterogeneity of the tumors. Moreover, Sciences, Chinese Academy of Medical Sciences and
drug resistance in pancreatic cancer is not only driven Peking Union Medical College. Cells were maintained at
by intrinsic tumor cell mechanisms but also by immune 37°C with 5% CO in high-glucose Dulbecco’s Modified
2
suppression within the tumor microenvironment (TME). Eagle Medium (DMEM, Invitrogen, USA), supplemented
5-8
Therefore, more effective therapeutic approaches are keen with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL
to address the challenges in improving pancreatic cancer penicillin (Hyclone, USA), and 100 μg/mL streptomycin
treatment outcomes. (Hyclone, USA). HHT was purchased from Xi’an Hao-
Xuan Bio-Tech Co., Ltd. (Shanxi, China). The HHT powder
Homoharringtonine (HHT), an alkaloid extracted from was dissolved in dimethyl sulfoxide to prepare a 20 mM
the herb Cephalotaxus, is a protein synthesis inhibitor that stock solution, which was further diluted to corresponding
inhibits the initial elongation step of translation by binding concentrations with DMEM complete medium for cellular
to the A-site of the ribosome, thereby inhibiting protein experiments.
synthesis. HHT has been approved by the U.S. Food and
9,10
Drug Administration for the treatment of chronic myeloid Healthy female C57BL/6 mice (6 – 8 weeks old) were
leukemia (CML) and acute myeloid leukemia (AML). 11,12 purchased and bred at the animal center of the Institute of
In recent years, studies on HHT have expanded to solid Basic Medical Sciences. All animal experiments reported
tumors. Several pioneering investigations have shown that were carried out in accordance with the guidelines of the
HHT exerts inhibitory effects on the growth of human committee on the Animal Care and Use of Institute of Basic
pancreatic cancer cells, such as inhibiting proliferation Medical Sciences, Chinese Academy of Medical Sciences
and growth of MIA-PaCa-2 and PANC-1 cells in a and Peking Union Medical College (Ethics Approval ID:
time- and dose-dependent manner. 13,14 In addition, HHT ACUC-A02-2023-015). Mice were euthanized through
has been shown to induce cell cycle arrest and enhance cervical dislocation following isoflurane anesthesia in
the anticancer activity of erlotinib in MIA-PaCa-2 and accordance with the 2020 AVMA Guidelines for the
Euthanasia of Animals, ensuring humane treatment. The
B×PC-3 cells. Nevertheless, despite its promising 20
15
potential in pancreatic cancer therapy, the underlying study adhered strictly to the ARRIVE guideline.
molecular mechanisms and therapeutic efficacy of HHT 2.2. Cell viability assay
require further investigation.
PANC-1, SW1990, Pan02-mCherry, and RAW264.7 cells
Previous studies on CML cells have indicated that HHT were seeded at a density of 5×10 cells per well and allowed
3
affects mitochondrial functions, particularly in oxygen to attach overnight. Subsequently, HHT concentrations
consumption rate (OCR). This implies the involvement of ranging from 0 to 400 nM were added to the cells. After 24
16
reactive oxygen species (ROS), which plays a crucial role in or 48 h of treatment, 10 μL of cell counting kit-8 (CCK-8)
maintaining cellular homeostasis in normal cells. However, reagent (Dojindo, Japan) was added, and the cells were
ROS dysregulation in cancer cells often leads to aberrant incubated for an additional 1 – 4 h at 37°C. Absorbance
cell signaling and uncontrolled proliferation. 17-19 Building was measured at 450 nm and 630 nm using a microplate
on this evidence, this study investigated the potential anti- reader (Thermofisher Scientific, USA). Cell viability was
tumor effects of HHT on PDAC cells and its underlying calculated as a percentage of the control, and the half-
mechanism of action, focusing on mitochondrial energy maximal inhibitory concentration (IC50) values were
metabolism in both tumor cells and stromal cells within the determined using GraphPad Prism software (GraphPad
TME. The results showed that HHT significantly inhibited Software, USA).
pancreatic cancer progression through significantly
elevating intracellular ROS levels over time, resulting 2.3. Cell proliferation assay
in the disruption of mitochondrial respiratory capacity, PANC-1 cells were washed with phosphate-buffered saline
impairment of energy metabolism, and cell cycle arrest. In (PBS) and resuspended in serum-free DMEM. Next,
addition, HHT alleviated the immunosuppressive effects carboxyfluorescein succinimidyl ester (CFSE, Dojindo,
within the TME by reducing the viability of macrophages. Japan) was added to the cell suspension at 5 μM, followed

Volume 4 Issue 1 (2025) 100 doi: 10.36922/td.7825