Terpinen-4-ol inhibits the proliferation and mobility of pancreatic cancer cells by downregulating Rho-associated coiled-coil containing protein kinase 2

Abstract

Terpinen-4-ol (T4O), a compound isolated from the seeds of turmeric, has exhibited anti-malignancy, anti-aging, and anti-inflammatory properties in previous studies. However, the specific effects and molecular mechanisms of T4O on pancreatic cancer (PC) cells remain largely unknown. In this study, we demonstrated that T4O markedly suppressed PC cell proliferation and colony formation in vitro and induced apoptosis. Similarly, T4O significantly inhibited the migration and invasion of PC cells in vitro. Through RNA sequencing, 858 differentially expressed genes (DEGs) were identified, which were enriched in the Rhodopsin (RHO)/ Ras homolog family member A (RHOA) signaling pathway. Rho-associated coiled-coil containing protein kinase 2 (ROCK2), a DEG enriched in the RHO/RHOA signaling pathway, was considered as a key target of T4O in PC cells; it was significantly reduced after T4O treatment, highly expressed in PC tissues, and negatively associated with patient outcome. Overexpression of ROCK2 significantly reduced the inhibitory effects of T4O on PC cell proliferation and mobility. Moreover, T4O inhibited cell proliferation in vivo and decreased the Ki-67, cell nuclear antigen, EMT markers, and ROCK2 expression. In conclusion, we consider that T4O can suppress the malignant biological behavior of PC by reducing the expression of ROCK2, thus contributing to PC therapy.

Keywords: Pancreatic cancer; ROCK2; mobility; proliferation; terpinen-4-ol.

Graphical abstract

Figure 1.

T4O inhibits PC cell proliferation in vitro. (a) AsPC-1 and PANC-1 cells were treated with different concentrations (0, 0.5, 1 and 2 μM) of T4O; CCK-8 assay was used to detect the proliferation in each group. (b) Colony formation assay was used to detect the colony formation of PC cells treated with different concentrations (0, 1, 2 and 4 μM) of T4O. *, P < 0.05; **, P < 0.01.

Figure 2.

T4O induces PC cell apoptosis in vitro. (a) AsPC-1 and PANC-1 cells were treated with different concentrations (0,0.5,1, and 2 μM) of T4O; flow cytometry was used to detect cell distribution in each group. (b) Western blotting was used to detect the expression of AsPC-1 and PANC-1 in cells treated with different concentrations (0,0.5,1, and 2 μM) of T4O. *, P < 0.05; **, P < 0.01.

Figure 3.

T4O suppresses the motility of PC cells in vitro. (a) Wound healing assays were used to determine the migration rate of PC cells treated with different concentrations (0,0.5,1, and 2 μM) of T4O. (b) Transwell assays were used to detect the number of invasive PC cells treated with different concentrations (0,0.5,1, and 2 μM) of T4O. (c) Western blotting was used to detect the expression of E-cadherin, vimentin, and N-cadherin in PC cells treated with T4O. *, P < 0.05; **, P < 0.01.

Figure 4.

ROCK2 is identified as a key target of T4O. (a-b) Differentially expressed genes (DEGs) in the PC cells treated with DMSO and T4O were identified. (c) KEGG analysis was performed to determine the pathways of enriched DEGs. (d) Gene enrichment plots showing a series of genes enriched in the RhoA/Rho kinase signaling pathway. (e) Western blot was used to detect the protein levels of ROCK2 in PC cells treated with different concentrations (0, 0.5, 1 and 2 μM) of T4O. (f) ROCK2 expression in PC tissues from patients in stages I–II and III–IV according to the data from ICGC. (g) qRT-PCR was used to detect the expression of ROCK2 in PC and adjacent tissues. (h) IHC was used to detect the expression of ROCK2 in PC and adjacent tissues. (i) Western blot was used to detect the expression of ROCK2 in HPDE, AsPC-1, and PANC-1 cells. (j) KM plot showing the OS and DFS in patients with low and high ROCK2 expression according to the data from ICGC database. *, P < 0.05; **, P < 0.01.

Figure 5.

Overexpression of ROCK2 reversed the inhibitory effects of T4O on proliferation of PC cells. PC cells were treated with DMSO + NC, T4O + NC, DMSO + ROCK2 plasmid, and T4O + ROCK2 plasmid, respectively. (a) Western blot was used to detect the expression of ROCK2, Bcl2, pro-caspase-3, cleave-caspase-3, N-cadherin, E-cadherin, and Vimentin in each group of PC cells. (b) CCK-8 assay was used to detect the proliferative rate of PC cells in each group in 24 and 48 h. (c) colony formation assays was used to detect the colony formation of PC cells in each group. **, P < 0.01.

Figure 6.

Overexpression of ROCK2 reversed the inhibitory effects of T4O on migration and invasion PC cells. PC cells were treated with DMSO + NC, T4O + NC, DMSO + ROCK2, and T4O + ROCK2 plasmids. (a) Wound healing assays were performed to determine the migration rate of the PC cells in each group. (b) Transwell assays were performed to detect the number of invasive PC cells in each group. **, P < 0.01.

Figure 7.

T4O represses PC cell proliferation in vivo. (a-b) Tumor volumes of tissues treated with DMSO and T4O. (c) Tumor weights of tissues treated with DMSO or T4O. (d) Expression of ROCK2, E-cadherin, N-cadherin, vimentin, PCNA, and KI67 in tumor tissues treated with DMSO and T4O. **, P < 0.01.