This study investigated the effect and mechanism of chloroquine on the growth of pancreatic neuroendocrine tumors (pNET). The research combined animal xenograft experiments with in vitro studies using the human pNET cell line QGP-1 and primary pNET cells isolated from surgical specimens.
Ten male BALB/c nude mice (6–8 weeks old) were subcutaneously implanted with QGP-1 cells in the right axilla to establish xenograft tumors. Mice were randomized by random number table to two groups (n = 5 per group): control (intraperitoneal saline once daily) and chloroquine (intraperitoneal 50 mg/kg once daily). Treatment continued for 4 weeks. At study end the mice were sacrificed and tumor long diameter and weight were compared. Tumor tissue was analyzed by immunohistochemistry for Ki-67 and by TUNEL fluorescence staining for apoptosis.
Tumors grew in both groups, confirming successful model establishment. The long diameter of transplanted tumors in the chloroquine-treated group was significantly smaller than in controls (P <0.05). No statistically significant differences in body weight between the groups were observed at the specified administration time points (days 1, 3, 5, 7, and 9 after administration; all P >0.05). The chloroquine group showed a lower proportion of Ki-67–positive cells and a higher proportion of TUNEL‑positive apoptotic cells compared with control (P <0.05).
Two cellular systems were used: primary pNET cells isolated from patients at the First Affiliated Hospital of Nanjing Medical University and the QGP-1 human pNET cell line. Primary cells were treated with chloroquine at 0, 10, 20, 40, 80, and 100 μmol/L (blank group received equivalent DMSO). QGP-1 cells were treated with 0, 20, 40, 60, 80, 100, and 160 μmol/L (blank group with DMSO). After 24, 48, and 72 hours of exposure, cell viability was measured by CCK-8 (A450 nm) and IC50 values were calculated.
For both primary pNET cells and QGP-1 cells, all tested chloroquine concentrations produced lower A450 viability values than blank at each time point (all P <0.05). The 24-hour IC50 was 67 μmol/L for primary pNET cells and 101 μmol/L for QGP-1 cells. Based on IC50 and considerations of sublethal cytotoxicity, subsequent functional assays used low, non‑highly cytotoxic concentrations: primary cells—blank, 10 μmol/L and 20 μmol/L; QGP-1—blank, 20 μmol/L and 40 μmol/L.
Proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) incorporation (proportion of EdU‑positive cells) and by plate colony formation (number of colonies). Migration and invasion were measured with Transwell assays (count of migrating and invading cells).
In primary pNET cells, treatment with 10 μmol/L and 20 μmol/L chloroquine reduced the proportion of EdU‑positive cells and the number of colonies, and decreased migrating and invading cell counts compared with blank (all P <0.05). The 20 μmol/L group produced greater reductions than the 10 μmol/L group and showed higher G0/G1 proportion and higher early and late apoptosis rates than the 10 μmol/L group (all P <0.05).
In QGP-1 cells, 20 μmol/L and 40 μmol/L chloroquine similarly reduced EdU‑positive cell proportion, colony numbers, and migration/invasion counts relative to blank, while increasing the proportion of cells in G0/G1 and raising early and late apoptosis rates.
Flow cytometry showed dose-dependent accumulation of cells in G0/G1 (or increased G1 proportion) and increases in early and late apoptosis rates with chloroquine treatment in both primary pNET cells and QGP-1 cells. These effects were statistically significant versus blank controls (P <0.05) and were greater at higher test concentrations within the sublethal ranges used.
The investigators performed transcriptome sequencing to screen differentially expressed genes and applied Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Western blotting was used to measure pathway‑related phosphorylated proteins—phosphorylated phosphatidylinositol 3-kinase (p‑PI3K), phosphorylated protein kinase B (p‑AKT), phosphorylated mammalian target of rapamycin (p‑mTOR)—and autophagy-related proteins ubiquitin-binding protein 62 (P62), Beclin‑1, and LC3‑II.
The abstract reports that these molecular assays were performed to explore mechanisms linking chloroquine exposure to tumor inhibition. However, the provided source excerpt does not include the detailed results or quantitative findings for the transcriptome enrichment or the Western blot analyses; those specific molecular outcomes were not reported in the available text.
Reported data indicate that chloroquine reduced tumor size in a QGP-1 xenograft model and inhibited proliferation, colony formation, migration and invasion while promoting G0/G1 cell-cycle arrest and apoptosis in both patient‑derived primary pNET cells and the QGP-1 cell line. The study conducted transcriptome and protein‑level assays aimed at identifying affected pathways, including the PI3K/AKT/mTOR axis and autophagy markers (P62, Beclin‑1, LC3‑II), but the detailed mechanistic results are not available in the provided abstract.
Limitations based on the available source: the excerpt does not provide full molecular result details, numerical Western blot data, statistical metrics beyond those summarized, or longer-term toxicity and safety assessments beyond short-term mouse body weight measurements. Additional methodological details (for example, randomization specifics, blinding, or full transcriptome results) were not reported in the provided text.
Within the limits of the reported data, chloroquine demonstrated anti‑tumor activity against pNET in vivo and in vitro, decreasing proliferative indices and increasing apoptosis. The authors evaluated transcriptomic changes and proteins related to the PI3K/AKT/mTOR pathway and autophagy, but the detailed molecular findings were not included in the accessible abstract. For complete mechanistic interpretation and potential translational implications, the full text should be consulted.