Selective cytotoxicity of the herbal substance acteoside against tumor cells and its mechanistic insights

Abstract

Natural products are characterized by extreme structural diversity and thus they offer a unique source for the identification of novel anti-tumor agents. Herein, we report that the herbal substance acteoside being isolated by advanced phytochemical methods from Lippia citriodora leaves showed enhanced cytotoxicity against metastatic tumor cells; acted in synergy with various cytotoxic agents and it sensitized chemoresistant cancer cells. Acteoside was not toxic in physiological cellular contexts, while it increased oxidative load, affected the activity of proteostatic modules and suppressed matrix metalloproteinases in tumor cell lines. Intraperitoneal or oral (via drinking water) administration of acteoside in a melanoma mouse model upregulated antioxidant responses in the tumors; yet, only intraperitoneal delivery suppressed tumor growth and induced anti-tumor-reactive immune responses. Mass-spectrometry identification/quantitation analyses revealed that intraperitoneal delivery of acteoside resulted in significantly higher, vs. oral administration, concentration of the compound in the plasma and tumors of treated mice, suggesting that its in vivo anti-tumor effect depends on the route of administration and the achieved concentration in the tumor. Finally, molecular modeling studies and enzymatic activity assays showed that acteoside inhibits protein kinase C. Conclusively, acteoside holds promise as a chemical scaffold for the development of novel anti-tumor agents.

Keywords: Acteoside; Cancer; Immunomodulation; Natural compound; Oxidative stress; Proteostasis.

Graphical abstract

Fig. 1

Acteoside is increasingly cytotoxic against metastatic cancer cellsvs. normal cells. (A₁) Chemical structure of acteoside. (A₂) Relative (%) cell survival of C5N and A5 cells after treatment with increasing concentrations of acteoside for 24 h. (B) Flow cytometry analysis of ROS levels in C5N and A5 cells after treatment with 500 μM acteoside for 24 h; values refer to mean (n = 3) fluorescence intensity. (C) Relative mRNA expression levels of genes involved in different proteostatic modules, antioxidant responses and metabolic pathways after treatment of C5N and A5 cells with 500 μM acteoside for 24 h. (D) Relative (%) levels of the CT-L/β5, C-L/β1 and T-L/β2 proteasomal peptidase (D₁) and lysosomal cathepsin B, L (D₂) activities in C5N and A5 cells following treatment (+ ) or not (-) with 500 μM acteoside for 24 h. (E) Gelatin zymography in A5 metastatic cancer cells showing MMP-9 and MMP-2 enzymatic activities after treatment (+ ) or not (-) with 500 μM acteoside for 24 h. Bars, ± SD (n ≥ 2). * , P < 0.05; **, P < 0.01.

Fig. 2

Acteoside induced proteostatic modules and exerted in vivo anti-tumor activity in a melanoma (B16.F1) grafted mouse model, likely via the activation of (among others) anti-tumor-reactive immune responses. (A) Relative (%) mRNA expression levels (in tumors of control and acteoside-treated mice) of genes involved in different proteostatic and antioxidant responses modules, as well as in metabolic pathways; acteoside was administered either intraperitoneally (IP) or via drinking water (OR). (B) Relative (%) proteasome (B₁) and cathepsin B, L (B₂) enzymatic activities in excised tumors of control (Con) and IP or OR acteoside-treated mice. (C) Average tumor volume in control mice (Con, n = 5); in mice injected every other day with 1 mg acteoside (IP) (n = 5) and in mice receiving 2.5 mg acteoside via drinking water (OR; n = 5). (D) Flow cytometry analysis of CD107 expression on splenocytes isolated from control and acteoside-treated mice. Splenocytes were used as effectors vs. the syngeneic B16.F1, YAC-1 and WEHI-164 target cells. Cells expressing CD107 are gated and the relative percentage is shown in each dot plot; representative dot plots per group with similar results are shown. Bars, ± SD; *P < 0.05; * *P < 0.01.

Fig. 3

Acteoside identification/quantification in mouse plasma and grafted tumors with the use of UHPLC-MS analyses. (A) Calibration curve of the analyte/IS vs. increasing concentrations of acteoside. (B) Representative LC-MS chromatogram of acteoside in mice plasma. (C) Representative MS spectrum of acteoside in treated mice plasma (C₁) and relative quantification (C₂) after OR or IP administration of the compound. (D) Quantification of acteoside in excised tumors after OR or IP administration of the compound in mice. Bars, ± SD (n ≥ 2). * , P < 0.05; * *, P < 0.01.

Fig. 4

Acteoside inhibits protein kinase C (PKC) activity. (A) Absorbance values showing purified PKC enzymatic activity in the absence (black bars) or presence (gray bars) of increasing concentrations of acteoside. (B) Relative (%) activity of immunoprecipitated PKC from C5N and A5 cells after exposure (+) or not (-) to 500 μM acteoside for 24 h. (C) Relative (%) PKC activity in isolated C5N, A5 and B16.F1 total cell lysates after treatment (+) or not (-) with acteoside (500 μM) for 24 h. (D) Representative immunoblots of protein samples from C5N and A5 (D₁), or from B16.F1 and B16.F10 cells (D₂) probed with an antibody against PKC. GAPDH probing was used as reference. (E) Four dominant binding poses of acteoside in the active site of PKC accounting for 86.6% of the total Boltzmann population as predicted by docking simulations. The protein is depicted in a ribbon representation colored according to its secondary structure, while the active site is shown as a semi-transparent molecular surface colored in gray; the ligand is depicted in a ball and stick model. Bars, ± SD (n ≥ 2). * , P < 0.05; * *, P < 0.01.