Yonsei Med J.  2018 Aug;59(6):727-735. 10.3349/ymj.2018.59.6.727.

Synergistic Anti-Cancer Effects of AKT and SRC Inhibition in Human Pancreatic Cancer Cells

• Ahn K
• O YM
• Ji YG
• Cho HJ
• Lee DH

• KMID: 2415531
• DOI: http://doi.org/10.3349/ymj.2018.59.6.727

Abstract

PURPOSE
To investigate the effect of combined inhibition of protein kinase B (AKT) and SRC on the growth and metastatic potential of human pancreatic cancer cells.
MATERIALS AND METHODS
AKT and SRC were inhibited using 10-DEBC and PP2, respectively. The expression of their messenger RNAs were down-regulated by specific small interfering RNA (siRNA). Changes in pancreatic cancer cell growth and metastatic potential were determined using a cell viability assay and a xenotransplant model of pancreatic cancer, as well as cell migration and invasion assays. Signal proteins were analyzed by Western blot.
RESULTS
The inhibitors 10-DEBC and PP2 suppressed cell proliferation in a dose-dependent fashion in pancreatic cancer cell lines MIA PaCa-2 and PANC-1. The simultaneous inhibition of AKT and SRC at low concentrations resulted in a significant suppression of cell proliferation. Knockdown of AKT2 and SRC using siRNAs also significantly decreased cell proliferation. In a pancreatic cancer model, combined treatment with 10-DEBC and PP2 also significantly suppressed the growth of pancreatic cancer. Application of 10-DEBC with PP2 significantly reduced the metastatic potential of pancreatic cancer cells by inhibiting migration and invasion. The combined inhibition suppressed the phosphorylation of mTOR and ERK in pancreatic cancer cells.
CONCLUSION
Combined targeting of AKT and SRC resulted in a synergistic efficacy against human pancreatic cancer growth and metastasis.

Keyword

AKT; SRC; pancreatic cancer; targeted therapy

MeSH Terms

Blotting, Western
Cell Line
Cell Movement
Cell Proliferation
Cell Survival
Humans*
Neoplasm Metastasis
Pancreatic Neoplasms*
Phosphorylation
Proto-Oncogene Proteins c-akt
RNA, Messenger
RNA, Small Interfering
Proto-Oncogene Proteins c-akt
RNA, Messenger
RNA, Small Interfering

Figure

• 

  Fig. 1 Effect of inhibitors against AKT and SRC on proliferation of human pancreatic cancer cells. (A and B) 10-DEBC and PP2 were added to MIA PaCa-2 (A) and PANC-1 (B) cells at different concentrations (0.1–300 µM; n≥6) for 72 h. Cell proliferation was determined via MTT assay. (C and D) After treatment with the inhibitors, 10-DEBC (1 µM, 3 µM) and PP2 (1 µM, 3 µM), or combination of 10-DEBC with PP2, the proliferation of MIA PaCa-2 (C) and PANC-1 (D) cells for 72 h was measured (n=9). Values represent mean±SEM. The response curve of nonlinear regression analysis fits the data using GraphPad Prism 5. *p<0.05 and **p<0.01 compared with control. AKT, protein kinase B.

• 

  Fig. 2 Effect of siRNAs against AKT and SRC in pancreatic cancer cell proliferation. After transfection with scrambled siRNA (SC) and siRNA against AKT1, AKT2, AKT3, or SRC (siAKT1, siAKT2, siAKT3, and siSRC, respectively), the proliferation of MIA PaCa-2 (A) and PANC-1 (B) cells for 48 h was measured (n=6). After simultaneous transfection with siAKT1 and siSRC, siAKT2 and siSRC, or siAKT3 and siSRC, the proliferation was measured. The SC was used as the control. Values are reported as mean±SEM. *p<0.05, **p<0.01, and ***p<0.001 compared with control. AKT, protein kinase B; siRNA, small interfering RNA.

• 

  Fig. 3 Effect of 10-DEBC and PP2 on pancreatic tumor growth in vivo. MIA PaCa-2 and PANC-1 cells were implanted in nude mice. Animals with established tumors were treated i.p. with phosphate-buffered saline, 10-DEBC (1 mg/kg), PP2 (1 mg/kg), or 10-DEBC with PP2. Administration started on day 0 and repeated on days 7, 14, and 21 for a total of four times (indicated by arrows). (A) Tumor size was measured once a week until day 28 (n=7). (B) Tumor weight was measured on day 28 after tumor tissue excision (n=7). (C) Representative photographs of tumor in each group are shown. Values are reported as mean±SEM. *p<0.05 and **p<0.01 compared with control. Vn and V0 indicates the average of tumor volumes on day n and the average of tumor volume on day 0, respectively (MIA PaCa-2 cells: left column, PANC-1 cells: right column).

• 

  Fig. 4 Effect of 10-DEBC and PP2 on migration and colony forming ability of pancreatic cancer cells. (A) After application of 10-DEBC (0.3 µM), PP2 (1 µM), and 10-DEBC combined with PP2, the migration capacities of MIA PaCa-2 (left) and PANC-1 (right) cells were monitored for 48 h using wound healing assay and expressed as percent of closed scratches (n=6). (B) Representative microscopic images show pancreatic cancer cells treated with 10-DEBC (0.3 µM), PP2 (1 µM), and 10-DEBC with PP2. (C) Following addition of 10-DEBC (0.3 µM), PP2 (1 µM), and 10-DEBC combined with PP2, colony formation by cultured pancreatic cancer cells in soft agar was observed. After 2 weeks, colonies were fixed by methanol, stained with 1% crystal violet, and counted to evaluate anchorage-independent growth of MIA PaCa-2 (left) and PANC-1 (right) cells (n=4). (D) Representative microscopic images show pancreatic cancer cells treated with 10-DEBC (0.3 µM), PP2 (1 µM), and 10-DEBC combined with PP2. Values are reported as mean±SEM. *p<0.05 and **p<0.01 compared with control.

• 

  Fig. 5 Effect of PP2 and 10-DEBC on mTOR and ERK in pancreatic cancer cells. MIA PaCa-2 cells were treated with PP2 (3 µM), 10-DEBC (3 µM), or PP2 combined with 10-DEBC for 1 h. The protein expression of mTOR and ERK and their phosphorylation was analyzed using Western blot. β-actin was used as a loading control. The molecular weights of mTOR, ERK, and β-actin were 289 kDa, 42/44 kDa, and 43 kDa, respectively. These experiments were performed in triplicate and the representative data are presented. mTOR, mammalian target of rapamycin; ERK, extracellular signal-regulated kinase.

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