Bakuchiol is extracted from
Gastric cancer is one of the most common malignant cancers globally. It is estimated that gastric cancer alone causes almost 10% of all cancer-associated mortality with a higher (10–12%) incidence rate in Asia and Europe (
Plant-derived natural products have been used as a source of medicinal agents useful to treat humans, and the hunt for novel, effective therapeutic compounds continues. Bakuchiol is a typical prenylated monoterpene phenolic compound separated from
Bakuchiol (
NUGC3 human gastric cancer cell lines were purchased from the Japanese Health Science Research Resources Bank (Osaka, Japan). The cells were cultured in RPMI-1640 medium and supplemented with heat-inactivated 10% FBS (v/v) and 1% penicillin and streptomycin in humidified conditions of 5% CO2 at 37°C.
To test the effect of bakuchiol on human gastric cancer cell viability, NUGC3 cells were seeded in a 96 well plate at a density of 6×104 cells/ml, then treated with 0, 20, 40, 60, 80, 100 or 120 µg/ml bakuchiol and incubated for 24 h at 37°C. The negative control cells were treated with DMSO. Following incubation, 0.5 g/ml MTT was added to each well and the plates were further incubated for 3 h at 37°C. The medium was then removed and DMSO was added to dissolve the formazan crystals formed. The absorbance was recorded at a 540 nm wavelength using an iMark (model 550) ELISA microplate absorbance reader (Bio-Rad Laboratories, Inc.).
NUGC3 cells were treated with 0, 50 and 100 µg/ml bakuchiol and incubated for 24 h at 37°C. The cell lines were collected and cleaned using cold PBS and centrifuged at 1,000 × g for 10 min at 37°C. Cold ethanol 70% (v/v) was used to fix the pellet at −20°C for 3 h, and the cells were then washed using PBS and 200 µl was transferred to a fresh tube. Muse Cell Cycle Assay kit solution (100 µl) was added to each well and incubated for 30 min in the dark at room temperature. The pellet was resuspended in 1 ml RPMI-1640 medium and 100 µl was transferred to a fresh tube for the analysis of apoptosis. Again, 100 µl of Muse Annexin V and Dead Cell Assay kit solution was added and incubated at room temperature for 30 min in the dark at 37°C and analyzed using the Muse cell analyzer (0500–3115) using Muse count and viable software (version 1.05.0) from EMD Millipore (Billerica, MA, USA).
NUGC3 cells were treated with 0, 50 and 100 µg/ml bakuchiol and incubated for 24 h at 37°C. A total of 1×104 cells were then washed with cold PBS at room temperature. Then the cells were fixed with 3.7% paraformaldehyde (1 ml) and 95% ethanol at room temperature for 10 min and followed by washing with PBS. The fixed cells were stained using DAPI (DAPI staining kit solution). The morphological nuclear changes were examined using fluorescence microscopy at ×400 magnification (Model-DMLS) from Leica Microsystems (GmbH, Wetzlar, Germany).
NUGC3 cells were treated with 0, 50 and 100 µg/ml bakuchiol at 37°C for 72 h and then lysed with NP-40 (1% w/w) ice cold radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), sodium deoxycholate (1% w/v), SDS (0.1% w/v), NaCl (0.15 M), EDTA (2 mM) sodium phosphate buffer (0.01 M, pH 7.2) and sodium fluoride (50 mM). The resultant cell lysates were centrifuged at 3,000 × g for 10 min at 37°C and total cellular protein concentration was determined by Bio-Rad Bradford protein assay method (Bio-Rad Laboratories, Inc.) (
The statistical analyses were performed using SPSS software (version 20.0; SPSS, Inc., Chicago, IL, USA). The results were expressed as the mean ± standard deviation of three independent experiments. Differences between the experimental groups were determined using one-way analysis of variance followed by Dunnett's multiple comparison post hoc test. P<0.05 was considered to indicate a statistically significant difference.
To determine the concentration of bakuchiol that inhibits cell viability, NUGC3 cells were treated with 0, 20, 40, 60, 80, 100 or 120 µg/ml bakuchiol for ~24 h, and cell viability was then measured by MTT assay. Following bakuchiol treatment and 24 h incubation, cell viability was inhibited in a concentration-dependent manner when compared with the control, without bakuchiol treatment (P<0.05;
Cell cycle distribution and the apoptotic cell population in bakuchiol-treated NUGC3 cells was determined using flow cytometry analysis. Bakuchiol treatment elevated the percentage of sub-G1 cells from 13% to 28 and 39% at 50 (P<0.05) and 100 µg/ml (P<0.01), respectively (
Treatment with bakuchiol (50 µg/ml; P<0.05 and 100 µg/ml; P<0.01) significantly increased the percentage of early apoptosis, late apoptosis and total apoptosis in NUGC3 cells in a concentration dependent manner compared with the control (
To confirm whether bakuchiol-induced apoptosis was caspase dependent, western blotting analysis was performed (
Cell proliferation and apoptosis are regulated by the PI3K/AKT and MAPK signaling pathways. As AKT activity is maintained by phosphorylation, the phosphorylation of PI3K/AKT and MAPK was analyzed using western blotting during bakuchiol-induced apoptosis in NUGC3 cells (
The aim of the present study was to investigate whether bakuchiol treatment induced gastric cancer cell death, and to further determine the underlying molecular mechanisms of bakuchiol-induced apoptosis in NUGC3 cells. Successful medicinal treatment using chemotherapeutic agents is primarily dependent on their potential to induce apoptosis in cancer cells (
The results of the MTT assay revealed that bakuchiol significantly reduced NUGC3 cell viability in a concentration dependent manner. Type I programmed cell death, or apoptosis, is the primary mechanism by which different anti-tumor and chemoprotective agents, inclusive of naturally derived products, exert anti-cancer properties (
To explore the involved molecular mechanisms, western blotting was performed and the results indicated that procaspase-3,6,8 and 9 expression levels were significantly decreased. The vital caspase is caspase-3, which activates PARP cleavage and results in the induction of the apoptosis process. Elevated cleaved caspase-3 expression concomitantly induced the cleavage of PARP. These data confirmed that bakuchiol induced apoptosis in a caspase-3 dependent manner. Mitochondrial apoptotic functions are regulated by apoptotic regulatory proteins of the Bcl-2 family. Cell viability and death were determined by the levels of Bcl-2 family pro-and anti-apoptotic proteins. The ratio of Bax and Bcl-xL has been demonstrated to be an apoptosis determining factor. In the present work, Bax protein expression levels remained the same while Bcl-xL levels were significantly downregulated, but following bakuchiol treatment the upregulation of the Bax:Bcl-xL ratio was observed in NUGC3 cells. When released from the mitochondria to the cytosol, cytochrome c has previously been observed to interact with apoptotic peptidase activating factor-1 and thereby lead to Bax/Bcl-xL ratio increment, thus activating caspase-3 and resulting in apoptosis (
To further evaluate the molecular mechanisms and involved signaling pathways in bakuchiol-induced apoptosis, PI3K/AKT and MAPK pathway phosphorylation levels were analyzed by western blotting. Bakuchiol inhibited levels of p-AKT, the downstream target of PI3K, which is known to control proliferation and cell apoptosis. Similar to the results of the present study, PI3K/AKT signaling pathway inhibition induces apoptosis in different cancer types (
In conclusion, bakuchiol was demonstrated to decrease cell viability and induce caspase-dependent apoptosis in NUGC3 gastric cancer cells. PI3K/AKT and MAPK signaling pathways triggered apoptosis following bakuchiol treatment in the NUGC3 gastric cancer cell line. To the best of our knowledge, the present study is the first to demonstrate the anticancer effect of bakuchiol treatment in NUGC3 cells. Therefore, bakuchiol may be an effective novel chemotherapeutic agent for treating human gastric cancer.
The authors would like to thank Xiangyang Central Hospital, Hubei University of Arts and Science for providing funding support to the present study (grant no. XCH2212-15).
The structure of bakuchiol.
Inhibitory effect of bakuchiol on NUGC3 cell viablity. (A) NUGC3 cell viability following treatment with bakuchiol, as determined by MTT assay. (B) Morphological changes in NUGC3 cells following treatment with 0, 50 or 100 µg/ml bakuchiol, examined under light microscopy (magnification, ×400). Data bearing different letters (a-d) were significantly different from each other (all P<0.05).
Cell cycle progression of NUGC3 cells treated with 0, 50 or 100 µg/ml bakuchiol for 24 h. The data represents the mean ± standard deviation of three independent experiments. *P<0.05; #P<0.01. vs. 0 µg/ml.
(A) Effect of 0, 50 or 100 µg/ml bakuchiol treatment on apoptosis of NUGC3 cells. (B) The data are expressed as the mean ± standard deviation of three independent experiments. (C) DAPI staining of NUGC3 cells with nuclear fragmentations indicated by white arrows, analyzed using a fluorescent microscope (magnification, ×400). *P<0.05; #P<0.01. vs. 0 µg/ml.
(A) Western blot analysis of caspase activation and PARP cleavage in NUGC3 cells treated with 0, 50 or 100 µg/ml bakuchiol for 24 h. Densitometric analyses of (B) procaspase-3, (C) procaspase-6, (D) procaspase-8, (E) procaspase-9, (F) cleaved caspase-3, (G) PARP and (H) Bax:Bcl-xL. *P<0.05; #P<0.01. vs. 0 µg/ml. PARP, poly (ADP-ribose) polymerase; Bax, B cell lymphoma-2 associated X, apoptosis regulator; Bcl-xL, B cell lymphoma-extra large.
(A) Effect of 0, 50 or 100 µg/ml bakuchiol treatment for 24 h on PI3K/AKT and MAPK signaling pathways in NUGC3 cells, analyzed by western blotting. Densitometric analyses of (B) p-Akt, (C) p-ERK1/2, (D) p-JNK and (E) p-p38. *P<0.05; #P<0.01. vs. 0 µg/ml. PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; MAPK, mitogen-activated protein kinase; p-, phosphorylated; ERK1/2, extracellular signal related kinase 1/2; JNK, c-Jun N-terminal kinase.