Amygdalin Exerts Antitumor Activity in Taxane-Resistant Prostate Cancer Cells

Abstract

Despite recent advances in the treatment of metastatic prostate cancer (PCa), resistance development after taxane treatments is inevitable, necessitating effective options to combat drug resistance. Previous studies indicated antitumoral properties of the natural compound amygdalin. However, whether amygdalin acts on drug-resistant tumor cells remains questionable. An in vitro study was performed to investigate the influence of amygdalin (10 mg/mL) on the growth of a panel of therapy-naïve and docetaxel- or cabazitaxel-resistant PCa cell lines (PC3, DU145, and LNCaP cells). Tumor growth, proliferation, clonal growth, and cell cycle progression were investigated. The cell cycle regulating proteins (phospho)cdk1, (phospho)cdk2, cyclin A, cyclin B, p21, and p27 and the mammalian target of rapamycin (mTOR) pathway proteins (phospho)Akt, (phospho)Raptor, and (phospho)Rictor as well as integrin β1 and the cytoskeletal proteins vimentin, ezrin, talin, and cytokeratin 8/18 were assessed. Furthermore, chemotactic activity and adhesion to extracellular matrix components were analyzed. Amygdalin dose-dependently inhibited tumor growth and reduced tumor clones in all (parental and resistant) PCa cell lines, accompanied by a G0/G1 phase accumulation. Cell cycle regulating proteins were significantly altered by amygdalin. A moderate influence of amygdalin on tumor cell adhesion and chemotaxis was observed as well, paralleled by modifications of cytoskeletal proteins and the integrin β1 expression level. Amygdalin may, therefore, block tumor growth and disseminative characteristics of taxane-resistant PCa cells. Further studies are warranted to determine amygdalin's value as an antitumor drug.

Keywords: amygdalin; cabazitaxel; cabozantinib; complementary/alternative medicine (CAM); docetaxel; prostate cancer; resistant cell lines.

Figure 1

Dose–response analysis of sensitive and drug-resistant prostate cancer cells. Growth of cabazitaxel-sensitive versus cabazitaxel-resistant cells has not been evaluated on PC3 cells. Error bars indicate standard deviation (SD). * indicates significant difference to the corresponding control, n = 5.

Figure 1

Dose–response analysis of sensitive and drug-resistant prostate cancer cells. Growth of cabazitaxel-sensitive versus cabazitaxel-resistant cells has not been evaluated on PC3 cells. Error bars indicate standard deviation (SD). * indicates significant difference to the corresponding control, n = 5.

Figure 2

Influence of amygdalin on the growth of parental, cabazitaxel- and docetaxel-resistant PC3, DU145, and LNCaP cell lines. Cell count is related to the 24 h value set to 100%. Error bars indicate standard deviation (SD), n = 5. * indicates significant difference to the corresponding non-treated control.

Figure 3

(A): BrdU uptake in sensitive and drug-resistant PC3, DU145, and LNCaP cells following amygdalin exposure for 24, 48, and 72 h. Values are given in percentage and are related to untreated controls, which were set to 100%. (B): Number of PC3, DU145, and LNCaP clones (drug-sensitive, cisplatin-resistant, and gemcitabine-resistant) exposed to amygdalin (+A), compared to the nontreated controls (−A). Error bars indicate standard deviation (SD), n = 3. * indicates significant difference to untreated controls. n.d.: not done.

Figure 4

Cell cycle distribution in drug-sensitive and drug-resistant PC3, DU145, and LNCaP cells following amygdalin exposure (+A) for 24 (all cell lines) and 72 h (LNCaP cells). Controls (−A) remained untreated. One representative of three separate experiments is shown (n = 3).

Figure 5

Up: Western blot of cell cycle and mTOR-related proteins from drug-sensitive and drug-resistant PC3, DU145, and LNCaP cells. Tumor cells received either amygdalin (+A) or cell culture medium alone (−A) for 24 h. β-actin served as the internal control. One representative from three separate experiments. n.d.: not done. Down: The ratio of protein intensity/β-actin intensity expressed as a percentage of the controls, set to 100%. Error bars indicate standard deviation, n = 3. * indicates significant difference to controls. n.d.: not done. Original blots see supplementary file S1.

Figure 5

Up: Western blot of cell cycle and mTOR-related proteins from drug-sensitive and drug-resistant PC3, DU145, and LNCaP cells. Tumor cells received either amygdalin (+A) or cell culture medium alone (−A) for 24 h. β-actin served as the internal control. One representative from three separate experiments. n.d.: not done. Down: The ratio of protein intensity/β-actin intensity expressed as a percentage of the controls, set to 100%. Error bars indicate standard deviation, n = 3. * indicates significant difference to controls. n.d.: not done. Original blots see supplementary file S1.

Figure 6

Adhesion of drug-sensitive and drug-resistant PC3 (A), DU145 (B), and LNCaP cells (C) to immobilized collagen, fibronectin, or matrigel. Tumor cells were either treated with amygdalin for 24 h (+A) or remained untreated (−A). (D) Effect of amygdalin (+A) on chemotactic migration of PC3 and DU145 cells. Controls were without amygdalin (−A). Bars indicate standard deviation (SD). * indicates significant difference to the corresponding control. n = 5. n.d.: not done.

Figure 7

Up: Western blot of cytoskeletal proteins and integrin β1 from drug-sensitive and drug-resistant PC3, DU145, and LNCaP cells. Tumor cells received either amygdalin (+A) or cell culture medium alone (−A) for 24 h. β-actin served as the internal control. One representative from three separate experiments. Down: The ratio of protein intensity/β-actin intensity expressed as a percentage of the controls, set to 100%. Error bars indicate standard deviation, n = 3. * indicates significant difference to controls. Original blots see supplementary file S1.