Andrographolide inhibits prostate cancer by targeting cell cycle regulators, CXCR3 and CXCR7 chemokine receptors

Abstract

Despite state of the art cancer diagnostics and therapies offered in clinic, prostate cancer (PCa) remains the second leading cause of cancer-related deaths. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of Andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on cell cycle phases in LNCaP, C4-2b and PC3 cells compared to retinoblastoma protein (RB(-/-)) and CDKN2A lacking DU-145 cells. G2/M transition was blocked in LNCaP, C4-2b and PC3 after AG treatment whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to differential activation of cell cycle regulators in these cell lines. Levels of cyclin A2 after AG treatment increased in all PCa cells line. Cyclin B1 levels increased in LNCaP and PC3, decreased in C4-2b and showed no difference in DU-145 cells after AG treatment. AG decreased cyclin E2 levels only in PC3 and DU-145 cells. It also altered Rb, H3, Wee1 and CDC2 phosphorylation in PCa cells. Intriguingly, AG reduced cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggests its potential use as a therapeutic and/or preventive agent for PCa.

Keywords: Andrographolide; CXCL11; CXCR3; CXCR7; Cell cycle; Cyclins; chemokine; chemokine receptor and prostate cancer.

Figure 1.

AG induces dose dependent death in PCa cell lines. Reduction in cell viability after AG treatment was assayed using MTT. Line graph represents 48 hr treatment data. Primary prostate epithelial cells were less sensitive to AG.

Figure 2.

Effect of AG on the status of cell cycle regulators in PCa cell lines. 20μM AG modulates phosphorylation of cell cycle checkpoints in PCa cells: LNCaP and C4-2b and, PC3 and DU-145. Protein samples were collected at 6 and 24 hr after AG treatment. Levels of GAPDH were used to verify equal protein loading. Histograms represent AG induced change in protein with respect to control at that time point.

Figure 3.

AG changes the cell cycle phase distribution of PCa cells. AG arrests LNCaP, C4-2b and PC3 cells in G2/M phase while DU-145 in G1 phase.

Figure 4.

AG affects chemokine receptor expression in PCa cells. Treatment with AG significantly reduced levels of CXCR3 and CXCR7 while CXCL11 is induced after 24 hr as assessed by western blot.

Figure 5.

CXCL11-CXCR3/7 axes affects viability of PCa cells. Blocking of CXCR3 or CXCR7 reduces the viability of PCa cells. Addition of CXCL11 also affects PCa cell viability. *, p value < 0.05; **, p value< 0.01 and ***, p value< 0.001 as compared to untreated.

Figure 6.

AG affects PCa cell migration via CXCR3-CXCL11 axis. Migration of all the 4 cell lines is drastically attenuated after AG treatment. Addition of CXCL11 increases while blocking of CXCR3 stunts migration of all the 4 cell lines at varied extent. AG treated cells do not show similar effect. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 when AG treated samples were compared to respective controls. ^$, p ≤ 0.05; ^$$, p ≤ 0.01; ^$$$, p ≤ 0.001 when compared to respective CXCL11 sample. ^#, p ≤ 0.05; ^##, p ≤ 0.01; ^###, p ≤ 0.001 when compared to untreated control. ^a, p ≤ 0.05; ^aa, p ≤ 0.01; ^aaa, p ≤ 0.001 when compared to only AG treated sample.