Nek2 siRNA therapy using a portal venous port-catheter system for liver metastasis in pancreatic cancer

Abstract

Nek2 (NIMA-related kinase 2) is a serine-threonine kinase and human homolog of the mitotic regulator NIMA of Aspergillus nidulan. We reported the efficiency of Nek2 siRNA in several cancer xenograft models using cholangiocarcinoma, breast cancer and colorectal cancer. Pancreatic cancer is difficult to treat due to its rapid progression and resistance to chemotherapy. Novel treatments are urgently required to improve survival in pancreatic cancer, and siRNA are a promising therapeutic option. However, finding an in vivo drug delivery system of siRNA remains a major problem for clinical application. In this study, the overexpression of Nek2 was identified in pancreatic cancer cell lines. Nek2 siRNA inhibited tumor growth in a subcutaneous xenograft mouse model of pancreatic cancer, prolonged the survival time in an intraperitoneal xenograft mouse model and efficiently prevented the progression of liver metastasis using a portal venous port-catheter system. Taken together, Nek2 is an effective therapeutic target in pancreatic cancer. An adequate delivery system is considered important in treating advanced pancreatic cancer, such as peritoneal dissemination and liver metastasis. Further investigations are required on the safety and side effects of the portal venous port-catheter system. We hope that Nek2 siRNA will be a novel therapeutic strategy for pancreatic cancer with liver metastasis and peritoneal dissemination.

Keywords: Liver metastasis; Nek2; pancreas cancer; portal venous port-catheter system; siRNA.

Figure 1

(a) Nek2 expression was examined in pancreatic cancer cell lines (Panc1, Mia Paca2, KLM1, KP4, PK45H, PK59, PK8 and PK9) and human foreskin fibroblast (HFF) cells by western blot analysis. β‐actin was used as an internal loading control. (b) Nek2 expression was analyzed in normal pancreatic tissues and cancer tissues by real time RT‐PCR. The expression levels in pancreatic cancer tissues are shown relative to the levels detected in normal tissues. *Statistically significant (P < 0.05 vs normal tissues by Student's t‐test). (c) Nek2 suppression by Nek2 siRNA (Nek2‐1 siRNA, Nek2‐2, Nek2‐3, Nek2‐4, Nek2‐5, Nek2‐6 and Nek2‐7) was analyzed in KLM‐1 cells (upper) and KP4 cells (lower) by western blot analysis. β‐actin was used as an internal loading control. (d) Cell proliferation was assessed in KLM‐1 cells using the WST‐1 cell proliferation assay. The cells were untreated, liposome only, transfected with control siRNA, or transfected with the Nek2 siRNA (Nek2‐1 siRNA, Nek2‐2, Nek2‐3, Nek2‐4, Nek2‐5, Nek2‐6 and Nek2‐7). The time points were 0 and 48 h after transfection. The data are shown relative to the zero time point. Each point represents the mean of six replicate wells. *Statistically significant (P < 0.05 vs control siRNA by Student's t‐test).

Figure 2

(a) Effect of Nek2 siRNA on tumor growth was examined in a subcutaneous xenograft mouse model. Photographs show the KLM1 xenograft tumor treated with Nek2‐1 siRNA and control siRNA at the end of the experiment. Graphs show the tumor volume of KLM xenograft tumors. Each bar in the graph represents the average tumor volume in each experimental group ± SE. *Statistically significant (P < 0.05 vs control siRNA by Student's t‐test). (b) Effect of intraperitoneal administration of Nek2 siRNA on an intraperitoneal xenograft mouse model. Photographs show peritoneal dissemination after treatment with Nek2‐1 siRNA and control siRNA. Total nodule number and weight were analyzed and compared between Nek2‐1 siRNA and control siRNA treated mice. Each bar in the graph represents the average tumor volume in each experimental group ± SE. *Statistically significant (P < 0.05 vs control siRNA by Student's t‐test). (c) Survival time was compared between Nek2‐1 siRNA and control siRNA treated mice.

Figure 3

(a) Cell motility was assessed in KP4 cells 48 h after treatment with Nek2‐1 siRNA using a scratch wound healing assay. Graphs show the distance of the scratch at six points. Each bar in the graph represents the average distance in each experimental group ± SE. *Statistically significant (P < 0.05 vs control siRNA by Student's t‐test). (b) Cell invasion was assessed in KP4 cells 18 h after treatment with Nek2‐1 siRNA with an invasion assay using modified Boyden chambers. Invaded cells were stained with HE. Graphs show the area of invaded cells in five randomly selected fields. *Statistically significant (P < 0.05 vs control siRNA by Student's t‐test) (c) Schema demonstrating the procedure for the liver xenograft rat model and portal venous port–catheter system. (d) Photographs show that fluorescently labeled Nek2 siRNA and liposome was injected in the liver using the portal venous port–catheter system. (e) Photographs show liver metastasis after treatment with Nek2‐1 siRNA or control siRNA using the portal venous port–catheter system. Graphs show the number and area of liver metastasis in each group. Each bar in the graph represents the average number and area of liver metastasis in each experimental group ± SE. *Statistically significant (P < 0.05 vs control siRNA by Student's t‐test).