Cucurbitacin I inhibits the proliferation of pancreatic cancer through the JAK2/STAT3 signalling pathway in vivo and in vitro

Abstract

Pancreatic cancer is one of the most aggressive solid malignancies, as it has a 5-year survival rate of less than 10%. The growth and invasion of pancreatic cancer cells into normal tissues and organs make resection and treatment difficult. Finding an effective chemotherapy drug for this disease is crucial. In this study, we selected the tetracyclic triterpenoid compound cucurbitacin I, which may be used as a potential therapeutic drug for treating pancreatic cancer. First, we found that cucurbitacin I inhibited pancreatic cancer proliferation in a dose-time dependent manner. Further studies have shown that cucurbitacin I blocks the cell cycle of pancreatic cancer in the G2/M phase and induces cell apoptosis. In addition, under the action of the compound, the invasion ability of cells was greatly reduced and markedly impaired the growth of pancreatic tumour xenografts in nude mice. Furthermore, the decrease in pancreatic cancer cell proliferation caused by cucurbitacin I appeared to involve JAK2/STAT3 signalling pathway inhibition, and the use of JAK2/STAT3 activators effectively restored the inhibition. In conclusion, our research may provide a basis for the further development of pancreatic cancer treatment drugs.

Keywords: Cucurbitacin I; JAK2/STAT3 signalling pathway; pancreatic cancer; proliferation.

Figure 1

CuI inhibited PDAC cell viability in a time-dose dependent manner. (A) Morphological changes of four PDAC cell lines (AsPC-1, BXPC-3, CFPAC-1, SW 1990) treated with different concentrations of CuI (0, 0.25, 0.5 and 1 µM), scale bar: 200 µm. (B) Chemical structure of CuI. (C) The cell viabilities were measured using the CCK-8. The line graph represents the percentage of viable cells in the control group.

Figure 2

CuI inhibited the proliferation of PDAC cells. (A-B) Detection by fluorescence microscopy of EdU (green) incorporated into the DNA of cultured BXPC-3 and CFPAC-1cells, scale bar: 200 µm. The nuclei were counter-stained with DAPI (blue). (C-D) The EdU positive rate of BXPC-3 and CFPAC-1 cells. (E) The colony formation assays of BXPC-3 and CFPAC-1 cells treated with different concentration (0-0.5 µM) of CuI. (F) The number of clones of BXPC-3 and CFPAC-1 cells.

Figure 3

CuI suppressed the migration and invasion of PDAC. (A) BXPC-3 cell migration under different concentrations of CuI treatment, scale bar: 200 µm. (B) Image J software was used to measure the distance of PDAC migration. (C) Transwell assays used to assess cell 3D-migration of BXPC-3, scale bar: 200 µm. (D) The area of migrating cells was counted by Image J software. (E) Transwell assays with matrix gel used to assess cell migration of BXPC-3, scale bar: 200 µm. (F) The area of invasion cells was counted by Image J software.

Figure 4

CuI arrested the PDAC cell cycle in the G2/M phase and induced apoptosis. (A) FACS analysis of PDAC cell cycle progression after CuI treatment. (B) The cell cycle distribution was calculated with Cell Quest Pro software. The cell cycle stagnates in the G2/M phase with the CuI dose. (C) Cell apoptosis was assessed with Annexin V-PI staining. (D) The group treated with 0.5 µM CuI showed the highest rate of apoptotic cells. (E) A decrease of Cyclin D1 and Cyclin A2 protein levels while increase of Cyclin B1 in CuI-treated PDAC cells. (F) A decrease of Caspase 3 and PARP1 protein levels while increase of cleaved Caspase 3 and cleaved PARP1 in CuI-treated PC cells.

Figure 5

CuI down-regulated JA2/STAT3 signalling pathway in PDAC cells. (A) CuI induced a decrease of p-JAK2 and p-STAT3 protein levels and does not influence the protein levels of T-JAK2 and T-STAT3 in CuI-treated PDAC cells. (B) Colivelin activated JAK2/STAT3 signalling pathway inhibited by CuI. (C-D) Colivelin significantly rescued the number of clones inhibited by CuI. (E) Colivelin significantly restored the number of cells inhibited by CuI.

Figure 6

CuI inhibited the growth of PDAC tumor xenografts in vivo. (A) Images of control group, low-dose (CuI 1mg/kg) and high-dose (CuI 1mg/kg) tumor-bearing mice. (B) Tumor masses from the control group, from the low-dose group and the high-dose group. (C) Measured the length and width every 3 days to calculate the volume of the tumor from day 0 to day 30 after injected two weeks. (D) Measured the weight of mice every 3 days. (E) CuI induced a decrease of p-JAK2 and p-STAT3 protein levels in mice tumor. (F) The expressions of PCNA in xenograft tumors were analyzed by immunohistochemistry.