[Effect and mechanism of chloroquine on supressing the growth of pancreatic neuroendocrine tumors].

Abstract

Objective: To investigate the effect and mechanism of chloroquine on the growth of pancreatic neuroendocrine tumors (pNET). Methods: This study was divided into animal experiments and cell experiments. (1) Animal experiments: ten male BALB/c nude mice, 6-8 weeks old, were selected. The suspension of QGP-1 cells (human pNET cell line) in the logarithmic growth phase was inoculated subcutaneously into the right axilla of nude mice to establish a subcutaneous xenograft tumor model. According to the random number table method, the mice were divided into a control group (intraperitoneal injection of physiological saline, once a day) and a chloroquine group (intraperitoneal injection of 50 mg/kg chloroquine, once a day), with 5 mice in each group. The treatment was administered continuously for 4 weeks. At the end of the 4th week, the mice were sacrificed, and the long diameter and weight of the transplanted tumors in each group were compared. The expression level of Ki-67 in tumor tissues was detected by immunohistochemistry (IHC) method, and tumor cell apoptosis was detected by TUNEL fluorescence staining. (2) Cell experiments: the cells used were human pNET cell line QGP-1 and pNET tissues (primary PNET cells) isolated from patients diagnosed with pNET at the First Affiliated Hospital of Nanjing Medical University who underwent surgical resection. Primary PNET cells were treated with 0, 10, 20, 40, 80, and 100 μmol/L chloroquine, and were respectively named the blank group [adding an equal volume of dimethyl sulfoxide (DMSO)] and 10, 20, 40, 60, 80, 100 μmol/L groups; QGP-1 cells were treated with 0, 20, 40, 60, 80, 100, and 160 μmol/L chloroquine, and were respectively named the blank group (adding an equal volume of DMSO) and 20, 40, 60, 80, 100, 160 μmol/L groups. After 24 hours of treatment, cell viability (absorbance A₄₅₀ nm value) was detected by cell counting kit-8 (CCK-8) method, and the half-maximal inhibitory concentration (IC50) was calculated. Based on the IC50 results, and combined with the long-term non-specific cytotoxicity, low-toxicity sub-lethal concentrations were selected for intervention in subsequent functional experiments. Cell proliferation ability was assessed by 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay (proportion of EdU-positive cells) and plate colony formation assay (number of cloned cell formations). Cell migration (number of migrating cells) and invasion ability (number of invading cells) were detected using Transwell method. Cell cycle (proportion of cells in G0 or G1 phase) and apoptosis (early and late cell apoptosis rates) were analyzed by flow cytometry. Differentially expressed genes were screened using transcriptome sequencing, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed. The expression levels of 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), key autophagy molecule Beclin-1 (Beclin1-1) and microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ)] were detected by Western blotting (WB). Results: (1) Animal experiments: tumors grew in both the control group and the chloroquine group, indicating successful modeling. The long diameter of the transplanted tumors in the chloroquine group was lower than that in the control group (P<0.05). There was no statistically significant difference in body weight between the chloroquine group and the control group at different administration times (days 1, 3, 5, 7, and 9 after administration) (all P>0.05). The proportion of Ki-67 positive cells in the chloroquine group was lower than that in the control group, while the proportion of TUNEL-positive cells (the number of apoptotic cells) was higher than that in the control group (P<0.05). (2) Cell experiments: after primary PNET cells were stimulated with different concentrations of chloroquine for 24, 48, and 72 h, the cell viability A₄₅₀ nm values of the 10, 20, 40, 60, 80, and 100 μmol/L groups at each time point were all lower than those of the blank group (all P<0.05); After QGP-1 cells were stimulated with different concentrations of chloroquine for 24, 48, and 72 h, the A₄₅₀ nm values of the 20, 40, 60, 80, 100, and 160 μmol/L groups at each time point were all lower than those of the blank group (all P<0.05). At 24 h of treatment, the IC50 values of primary PNET cells and QGP-1 cells were 67 and 101 μmol/L, respectively; therefore, in subsequent experiments, the blank group, 10 μmol/L group, and 20 μmol/L group were selected for primary PNET cells, and the blank group, 20 μmol/L group, and 40 μmol/L group were selected for QGP-1 cells. The proportion of EdU-positive cells, the number of cloned cell formations, and the number of migrating and invading cells in the 10 μmol/L and 20 μmol/L primary PNET cell groups were all less than those in the blank group, while the proportion of cells in the G1 phase and the apoptosis rate were all higher than those in the blank group (all P<0.05); additionally, the proportion of EdU-positive cells, the number of cloned cell formations, and the number of migrating and invading cells in the 20 μmol/L group of primary PNET cell were all less than those in the 10 μmol/L group, while the proportion of cells in the G0 or G1 phase and the (early and late) cell apoptosis rates were all higher than those in the 10 μmol/L group (all P<0.05). The proportion of EdU-positive proliferating cells, the number of cloned cell formations, and the number of migrating and invading cells in the 20 μmol/L and 40 μmol/L group of QGP-1 cell were all less than those in the blank group, while the proportion of cells in the G0 or G1 phase and the (early and late) cell apoptosis rates were all higher than those in the blank group (all P<0.05); additionally, the proportion of EdU-positive proliferating cells, the number of cloned formations, and the number of migrating and invading cells in the 40 μmol/L group of QGP-1 cell were all less than those in the 20 μmol/L group, while the proportion of cells in the G0 or G1 phase and the (early and late) cell apoptosis rates were all higher than those in the 20 μmol/L group (all P<0.05). Transcriptome sequencing analysis showed that differentially expressed genes were enriched in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway; WB method results showed that the relative expression levels of phosphorylated proteins such as p-mTOR, p-PI3K, and p-AKT in the 20 μmol/L group of QGP-1 cells were lower than those in the blank group (all P<0.05); the relative expression levels of the autophagy substrate P62 and the autophagosome marker LC3-Ⅱ protein were higher than those in the blank group, while Beclin-1 was lower than that in the blank group (all P<0.05). Conclusion: Chloroquine may inhibit the occurrence and development of pNET by suppressing the phosphorylation activation of the PI3K/AKT/mTOR signaling pathway and blocking autophagy.