Traditional Herbal Formula Taeeumjowi-Tang (TJ001) Inhibits p53-Mutant Prostate Cancer Cells Growth by Activating AMPK-Dependent Pathway

Abstract

Dysregulated lipid metabolism is a prominent feature of prostate cancers (PCas); several enzymes involved in lipid accumulation are highly expressed. Here, we elucidated efficacy of TJ001, a traditional herbal decoction, in inhibiting de novo lipogenesis. TJ001 had significant cytotoxicity against DU145 but not PC3 and LNCaP cells and, similarly, TJ001 markedly AMPK phosphorylation only in DU145 cells. This was accompanied by the downregulation of phosphorylated-acetyl coenzyme A carboxylase (ACC) expression and sterol regulatory element-binding protein 1 (SREBP1) proteolytic cleavage, thereby inhibiting its role as a transcription factor to induce lipid biosynthesis. When Oil Red O staining was performed, it is reflected in the reduction of lipid droplets (LDs). TJ001 also induced G₁/S cell cycle arrest via a cell cycle inhibitor (CKI) p21^WAF1/CIP1 upregulation. Although p53 proteins remained unchanged, both cyclin E and cyclin D1 were decreased. Moreover, TJ001 suppressed the mammalian target of rapamycin (mTOR) signaling pathway. Generally, the prolonged G₁/S phase arrest accompanies apoptosis, but TJ001 failed to work as a trigger apoptosis in DU145 cells. We showed that mutant p53 proteins were required for the survival of DU145 cells. In presence of TJ001, inhibition of endogenous mutant p53 by RNAi led to cell viability reduction and induction of the p-AMPK/AMPK ratio. In addition, it induced apoptotic cell death in DU145 cells. At the cellular level, induction of PARP, caspase-3, and caspase-9 cleavages was observed, and caspase-3 activity was increased in the p53 knockdown cells treated with TJ001. Taken together, we demonstrated that TJ001 inhibited cell growth in DU145 prostate cancer cells as indicated by blocking lipogenesis and induction in G₁/S cell cycle arrest. In addition, we may provide an evidence that mutant p53 protein has potential role as an oncogenic action in DU145 cells. Collectively, the combination of mutant p53 targeting and TJ001 treatment resulted in decreased cell growth in DU145 cells.

Figure 1

TJ001 inhibits cell growth in prostate cancer cells. (a) The regulation of prostate cancer (p53 mutant DU145, p53 null PC3, and p53 wild-type LNCaP) cells proliferation by TJ001. Cell viability was measured by the MTT assay; optical density of MTT solution was measured at 590 nm. Data represent the average of three independent experiments [error bars are standard deviation (SD) (∗∗∗p < 0.001)]. (b) Cell viability after TJ001 treatment in normal cells. (c) Clonogenic ability of DU145, PC-3 and LNCaP cells after TJ001 treatment. Cells were treated with or without 200 μg/mL TJ001 for 7 days. The formed colonies were stained using 0.25% crystal violet. [Left panel; clonogenic plates with 0 μg/mL, right panel; clonogenic plates with 200 μg/mL TJ001.] (d) The graph showed the number of colonies in (c). Error bar represents SD (∗p < 0.05).

Figure 2

Inhibitory effects of TJ001 on lipid accumulation in DU145 cells. Cells were incubated for 48 h with or without TJ001 (200 μg/mL). (a) The content of ATP was measured using a commercial kit (Promega, USA). Data are presented as the mean ± SD (∗P < 0.05 compared with the control). We analyzed (b) the expression of lipid metabolism-related proteins, (c) the effects of compound C (c.c) on phosphorylated AMPK (p-AMPK). (d) Lipid accumulation was visualized using an Olympus CKX41 inverted microscope at ×300 magnification [left panel; Oil Red O stained cells with 0 μg/mL, right panel; with 200 μg/mL TJ001].

Figure 3

TJ001 induces G1/S phase arrest in DU145 cells. Cell cycle distribution of DU145 cells measured by flow cytometry. (a) Cells were incubated with TJ001 (200 μg/mL) treatment for 48 h. All floating and attached cells were harvested and fixed in 95% ethanol. The stained cells with PI were performed to examine cell cycle progression (b) The graphs showed a cell cycle distribution in DU145 prostate cancer cells. Data represents mean ± SD (∗∗ p< 0.01) (c) CKI (Cyclin kinase inhibitor) and cyclin proteins, involved with G1/S phase arrest, were performed. (d) Total and phosphorylated forms of protein synthesis-related proteins in mTOR signaling pathway were detected using immunoblotting assay.

Figure 4

TJ001 had no effects on apoptosis in DU145 cells. (a) Cells (1X10²) were grown with or without TJ001 (200 μg/mL) in 60pi dish for 48 h. Doxorubicin (DOX) was also used as a positive control. Harvested cells were stained with annexin-V-FITC for detecting apoptosis, then were stained with 7-AAD for detecting DNA contents for 15 min each. Cell apoptosis was determined by FACS analysis. (b) Apoptosis-regulated protein levels were evaluated after TJ001 (200 μg/mL) treatment. Anti-β-actin was a loading control.

Figure 5

Effects of mutant p53 knockdown with TJ001 treatment in DU145 cells. After cells were transfected with p53 siRNA or control siRNA, and then cells were treated with TJ001. (a) The cell viability was evaluated by WST-1 assay. (∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001). The total lysates were assessed by western blot analyses. Following to transfection, (b) expression of p-AMPK/AMPK ratio, (c) levels of cell cycle-related proteins expression and (d) PARP, capsase-3, capsase-8, and capsase-9 were measured. (e) Quantitative analysis of caspase-3 activity in vitro. Cell proteins were diluted in Cell Lysis Buffer and assayed for caspase-3 activity using DEVD-pNA as substrate. Results were measured at 405 nm wavelength (∗∗∗p<0.001).