Biological and physical approaches on the role of piplartine (piperlongumine) in cancer

Abstract

Chronic inflammation provides a favorable microenvironment for tumorigenesis, which opens opportunities for targeting cancer development and progression. Piplartine (PL) is a biologically active alkaloid from long peppers that exhibits anti-inflammatory and antitumor activity. In the present study, we investigated the physical and chemical interactions of PL with anti-inflammatory compounds and their effects on cell proliferation and migration and on the gene expression of inflammatory mediators. Molecular docking data and physicochemical analysis suggested that PL shows potential interactions with a peptide of annexin A1 (ANXA1), an endogenous anti-inflammatory mediator with therapeutic potential in cancer. Treatment of neoplastic cells with PL alone or with annexin A1 mimic peptide reduced cell proliferation and viability and modulated the expression of MCP-1 chemokine, IL-8 cytokine and genes involved in inflammatory processes. The results also suggested an inhibitory effect of PL on tubulin expression. In addition, PL apparently had no influence on cell migration and invasion at the concentration tested. Considering the role of inflammation in the context of promoting tumor initiation, the present study shows the potential of piplartine as a therapeutic immunomodulator for cancer prevention and progression.

Figure 1

The docked position of PL onto the Ac_2-26 peptide. (a) Secondary structure of annexin A1 is represented in orange, Ac_2-26 peptide in dark blue and PL as sticks. (b, c) As shown, the Ac_2-26 peptide binds to PL via two hydrogen bonding interactions (dotted lines) at lysine 9 (in green) but not at tryptophan 12 (in yellow). Data were taken, with permission, from experiments performed by the author Henrique T, and figure printed in. Figure was generated by Pymol system version 2.0 (https://pymol.org/).

Figure 2

Energy contributions to the interaction between Ac_2-26 and PL at different excitation wavelengths (280 nm and 295 nm). Monitoring the microenvironment of the interaction around the aromatic tryptophan residue. ΔG = Gibbs free energy changes, ΔH = enthalpy changes, ΔS = entropy changes, T = temperature. Data were taken, with permission, from experiments performed by the authors Contessoto NSA and Cornélio ML, and figure printed in.

Figure 3

PL and Ac_2-26 treatments decrease the number of viable HEP-2 cells, but have a low effect on HUVEC viability. MTS assay was used to determine proliferation and viability of neoplastic and normal cells treated with PL or Ac_2-26 alone or in combination for 24 h, 48 h and 72 h. An equivalent volume of vehicle (final concentration in culture medium = 10 µg/mL) without PL or Ac_2-26 was added to the DMSO control group, and no DMSO/PL/Ac_2-26 to the negative control. Assays were carried out in triplicate, and experiments were performed two times (ANOVA *p < 0.05; **p < 0.001).

Figure 4

PL apparently has no effect on migration and invasion of HUVEC and HEp-2 cells. (a) Representative images of cell migration and (b) invasion assays performed on cells treated with PL alone or in combination with Ac_2-26 (cells stained by DAPI) (left panels). Quantitative analysis of the number of migrated and invasive cells relative to untreated controls (right panels). An equivalent volume of vehicle (final concentration in culture medium = 10 µg/mL) without PL or Ac_2-26 was added to the DMSO control group, and no DMSO/PL/Ac_2-26 was added to the negative control. Assays were carried out in triplicate (ANOVA *p < 0.05; **p < 0.001).

Figure 5

PL inhibits α-tubulin expression. Western blotting analysis of HUVECs treated with PL alone or in combination with Ac_2-26 and/or LPS shows that PL inhibits α-tubulin (50 kDa) expression. β-actin (42 kDa) was used as an endogenous control. No LPS/PL/Ac_2-26 was added to the negative control. Fold change relative to control was calculated using the α-tubulin: β-actin ratio. Reproduced with permission from. The vertical streaks on the blot images were probably caused by a contaminant on the scanning lamp/lens assembly glass.

Figure 6

PL modulates chemokine and cytokine expression. ELISA analysis of proinflammatory cytokines MCP-1 (a, d), IL-8 (b, e), and IL-1β (c, f) from culture supernatants of HUVECs treated with LPS, Ac_2-26 and/or PL for 24 and 72 h. No LPS/PL/Ac_2-26 was added to the negative control. Results represent the mean ± SEM from of three independent assays (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus untreated control; ^&p < 0.05, ^&&p < 0.05 versus Ac_2-26. Reproduced with permission from.