Antiproliferative and antimetabolic effects behind the anticancer property of fermented wheat germ extract

Abstract

Background: Fermented wheat germ extract (FWGE) sold under the trade name Avemar exhibits anticancer activity in vitro and in vivo. Its mechanisms of action are divided into antiproliferative and antimetabolic effects. Its influcence on cancer cell metabolism needs further investigation. One objective of this study, therefore, was to further elucidate the antimetabolic action of FWGE. The anticancer compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ) is the major bioactive compound in FWGE and is probably responsible for its anticancer activity. The second objective of this study was to compare the antiproliferative properties in vitro of FWGE and the DMBQ compound.

Methods: The IC50 values of FWGE were determined for nine human cancer cell lines after 24 h of culture. The DMBQ compound was used at a concentration of 24 μmol/l, which is equal to the molar concentration of DMBQ in FWGE. Cell viability, cell cycle, cellular redox state, glucose consumption, lactic acid production, cellular ATP levels, and the NADH/NAD(+) ratio were measured.

Results: The mean IC50 value of FWGE for the nine human cancer cell lines tested was 10 mg/ml. Both FWGE (10 mg/ml) and the DMBQ compound (24 μmol/l) induced massive cell damage within 24 h after starting treatment, with changes in the cellular redox state secondary to formation of intracellular reactive oxygen species. Unlike the DMBQ compound, which was only cytotoxic, FWGE exhibited cytostatic and growth delay effects in addition to cytotoxicity. Both cytostatic and growth delay effects were linked to impaired glucose utilization which influenced the cell cycle, cellular ATP levels, and the NADH/NAD(+) ratio. The growth delay effect in response to FWGE treatment led to induction of autophagy.

Conclusions: FWGE and the DMBQ compound both induced oxidative stress-promoted cytotoxicity. In addition, FWGE exhibited cytostatic and growth delay effects associated with impaired glucose utilization which led to autophagy, a possible previously unknown mechanism behind the influence of FWGE on cancer cell metabolism.

Keywords: Autophagy; Benzoquinone; Cancer cells; Cytostatic; Cytotoxicity; FWGE; Reactive oxygen species.

Fig. 1

Antiproliferative properties of FWGE and DMBQ. The effects of FWGE (mean IC₅₀: 10 mg/ml) and DMBQ (24 μmol/l; equal to the DMBQ concentration in FWGE) on cancer cell viability (a). Intracellular DCF fluorescence signals indicating intracellular ROS formation after 12 h (BxPC-3 cells) and 24 h (23132/87 and HRT-18 cells) of culture (b). The dashed line indicates the relative initial cell count at the start of treatment. For this, the seeded cells were stained with crystal violet directly after their adherence and the absorbance was normalized to 100 %. By definition, a cytotoxic effect was a reduction in initial viable cell count >15 %, a cytostatic effect a change in initial cell count of ±15 %, and a delayed growth effect an increase in the initial cell count >15 %. Ascorbic acid (2.4 mmol/l) was used to activate DMBQ [16]. Ascorbic acid had no influence on cancer cell viability or FWGE (not shown). Results are shown as mean ± standard deviation and representative for at least three independent experiments performed in triplicate. **P < 0.01, ***P < 0.001 in comparison to untreated control cells. RFU, relative fluorescence units

Fig. 2

FWGE-influenced cancer cell metabolism. Lactic acid production (mmol/l for 10⁴ cells) and glucose consumption (mmol/l for 10⁴ cells) by 23132/87 cells (a, c) and HRT-18 cells (b, d) with and without FWGE treatment for the incubation times indicated. Cellular ATP content (pg/10⁶ cells) and the NADH/NAD⁺ ratio in 23132/87 cells (e) and HRT-18 cells (f) with and without FWGE treatment after 24 h of culture. Results are shown as mean ± standard deviation for three independent experiments.***P < 0.001 to FWGE-untreated cells

Fig. 3

Viability of HRT-18 and 23132/87 cells continuously cultured with FWGE. Incubation of 23132/87 and HRT-18 cells with 10 mg/ml FWGE was cytotoxic after 72 h of continuous culture for 23132/87 cells and after 120 h for HRT-18 cells. Cells were incubated in medium with 10 % (v/v) fetal calf serum and cell viability was determined by crystal violet staining at the culture times indicated. The dashed line indicates the relative initial cell count at the start of treatment. For this, the seeded cells were stained with crystal violet directly after their adherence and the absorbance was normalized to 100 %. By definition, a cytotoxic effect was a reduction in initial viable cell count >15 %, a cytostatic effect a change in initial cell count ±15 % and a delayed growth effect an increase in the initial cell count >15 %. Results are shown as mean ± standard deviation for three independent experiments, each performed in triplicate for each time point. ***P < 0.001 in comparison to 24 h

Fig. 4

FWGE-induced autophagic activity in the colon carcinoma cell line HRT-18. FWGE-treated HRT-18 cells showing intracellular vacuoles 24 h after start of incubation. The vacuoles increased in size with increasing incubation time (a). Presence of endogenous LC3-II in HRT-18 cells with and without FWGE treatment at different incubation times (b). LC3-I (approximately 17 kDa) is the cytosolic form of LC3, which is converted into the active, membrane-bound form LC3-II (approximately 14–15 kDa) during the autophagy process. β-actin was used as a loading control (42 kDa). The shift from LC3-I to LC3-II is evident following FWGE treatment. Western blot results are shown for one trial representative of three independent experiments (c). The LC3-II/LC3-I ratio was calculated based on densitometry analysis (ImageJ 1.3.4 s downloaded from the National Institutes of Health (NIH), Bethesda, MD, USA) of LC3-I and LC3-II bands for three independent experiments (***P < 0.001, *P < 0.05 in comparison to FWEG-untreated cells)