Contributed equally
Cancer is one of the major issues affecting public health and is considered the leading cause of mortality worldwide (
Apoptosis is a type of programmed cell death accompanied by cell membrane contraction, nucleus fragmentation, chromatin condensation and chromosome DNA breakage (
TCM has long been widely used for the prevention and treatment of various diseases, including cancer in China and other Asian countries. TCM has attracted increasing attention and has become an emerging field for anticancer drug discoveries and development due to its wide availability in nature and the fact that it can be easily obtained; moreover, some active components isolated and extracted from TCM have exhibited significant antitumor activity with less side-effects compared to commonly used chemotherapeutic agents in cancer therapy (
The present study investigated the
The seeds of AKH (cat. no. T000500063) were purchased from Sichuan Hongpu Pharmaceutical Co., Ltd. A total of 10 kg dehydrated powdered seeds of AKH were extracted with 95% EtOH (Chengdu Chron Chemicals Co., Ltd.) for 48 h at room temperature. The EtOH extraction was concentrated with a rotary evaporator under 50°C under reduced pressure as previously described (
3MA was purchased from MilliporeSigma. RPMI-1640 medium, fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) were purchased from Gibco; Thermo Fisher Scientific, Inc. The Hoechst 33342/PI kit and Baf-A1 were purchased from Beijing Solarbio Science & Technology Co., Ltd. The Cell Counting Kit (CCK)-8 kit, Ad-GFP-LC3B and actin mouse monoclonal antibody (cat. no. AF0003) were purchased from the Beyotime Institute of Biotechnology. The Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences. The pro-caspase-3 (cat. no. ab32150), cleaved caspase-3 (cat. no. ab32042), caspase-8 (cat. no. ab32397), caspase-9 (cat. no. ab32068), PARP (cat. no, ab32138) and cleaved PARP (cat. no. ab32561) antibodies were purchased from Abcam. LC3B (cat. no. 2775S), Beclin-1 (cat. no. 3738S), AMPK (cat. no. 2532S), phosphorylated (p-)AMPK at Ser 485 (cat. no. 4184S), Akt (cat. no. 9272S), p-Akt at Ser473 (cat. no. 9271S), mTOR (cat. no. 2972S), p-mTOR at Ser2448 (cat. no. 2971S), p70S6K (cat. no. 9202S) and p-p70S6K at Thr389 (cat. no. 9205S) antibodies were purchased from Cell Signaling Technology, Inc.
The human lung cancer A549 (cat. no. CCL-185), breast cancer MDA-MB-468 (cat. no. HTB-132), cervical cancer HeLa (cat. no. CCL-2), glioblastoma of unknown origin U87 (cat. no. HTB-14) and colon cancer HCT-116 (cat. no. CCL-247) cell lines were purchased from the American Type Culture Collection (ATCC). The human melanoma A875 (cat. no. CL-0255) and normal hepatic stellate LX-2 (cat. no. CL-0560) cell lines were purchased from Procell Life Science & Technology. The human pancreatic cancer Panc-28 cell line was kindly provided by Dr Joshua Liao (University of Minnesota, Austin, MN, USA). All cancer cells were cultured in RPMI-1640 medium, whereas the LX-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin and maintained in an atmosphere of 5% carbon dioxide (CO2) at 37°C and renewed with new medium every 3–5 days. The LX-2 cells were analyzed by short tandem repeat profiling, cell morphology and karyotyping assay. All cell lines were verified on the Cellosaurus database and it was confirmed that they were not considered problematic for use.
The effect of AKH on the viability of various cancer cells and normal hepatic stellate LX-2 cells was evaluated by CCK-8 assay. Briefly, cells (>90% confluency, 5×103 cells/well) were seeded in 96-well culture plates. Following overnight incubation, the cells were treated with the solvent solution (vehicle control) or various concentrations of AKH (50, 100, 150, 200, 300 and 400 µg/ml) for 48 h, respectively. For the study of time-dependent effects of AKH on cell growth inhibition, the Panc-28 and A549 cells (the two most sensitive cells to AKH treatment (
Apoptotic cancer cells were initially detected using the Hoechst 33342/PI double stain apoptosis detection kit (Beijing Solarbio Science & Technology Co., Ltd.) following the manufacturer's instructions. Briefly, the Panc-28 or A549 cells (>90% confluency) were seeded in six-well plates at a density of 3×105 cells/well. The cells were treated with the vehicle control or AKH at 150, 200 and 250 µg/ml for 48 h following 24 h of culture. The medium was then removed, and the cells were washed with PBS for three times in 15 min (5 min each). Finally, the cells were stained with Hoechst 33342/PI kit in the dark for 30 min at room temperature and apoptotic cells were observed under a Leica DM6 B fluorescence microscope (Leica Microsystems GmbH). All experiments were performed for at least three times in triplicate.
The detection of the apoptosis of Panc-28 and A549 cells was also performed using flow cytometric analysis with the Annexin V-FITC apoptosis detection kit according to the manufacturer's protocol as described in a previous study by the authors (
To detect the formation of autophagosomes, the Panc-28 and A549 cells were transfected with Ad-GFP-LC3B (Beyotime Institute of Biotechnology) following the manufacturer's instructions. Briefly, the cells (>90% confluency) were seeded in a 24-well plate at a density of 5×104 cells/well and transfected with Ad-GFP-LC3B at a multiplicity of 40 with 2×106 plaque forming units (pfu) adenovirus for 24 h at room temperature. The cells were then treated with medium (control) or AKH (250 µg/ml) for 24 h, respectively. They were then fixed with 4% polyoxymethylene and observed under a Leica DM6 B fluorescence microscope at ×40 magnification (Leica Microsystems GmbH). All experiments were performed for at least three times in triplicate.
The Panc-28 and A549 cells (>90% confluency) were seeded in six-well plates at a density of 5×105 cells/well. Following 24 h of incubation, the cells were treated with the solvent solution or AKH at 150, 200 and 250 µg/ml for 48 h. The cells were then collected by centrifugation at 1,500 × g at room temperature for 5 min and lysed in ice-cold RIPA lysate buffer as described in a previous study by the authors (
Female athymic Balb/C nude mice (6–8 weeks old; body weight, 18–20 g) were purchased from Chengdu Dasuo Laboratory animal Co., Ltd. and housed in a specific pathogen-free (SPF) facility with a constant laboratory condition of a 12-h light/dark cycle and provided with food and water
All animal experiments were approved (permit no. 201802-112) by the Institutional Animal Care and Use Committee of Southwest Medical University (Luzhou, China) and strictly followed the guidelines for the investigation of experimental pain in conscious animals for improving animal welfare to minimize animal suffering (
LC-MS analysis of the composition of AKH was performed using a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu Corp.). The samples of AKH were separated on an Agilent Eclipse plus C18 column (100×2.1 mm i.d. 1.8 µM particle size; Agilent Technologies, Inc.). The separation process was followed the gradient elution procedure. Chromatographic analysis was used the mobile phase A, which was composed of acetonitrile modified with 0.5% formic acid, while the mobile phase B was composed of water modified with 0.5% formic acid. The linear gradient was as A:B=40–90% for 24 min. The flow rate was 0.2 ml/min and the column temperature was 30°C. The injection volume was 5 µl. For mass detection, the following parameters were used for analytical MS: Nozzle voltage, 4.5 KV (+)/-3.5 KV (−) in the detection modes of positive ion and negative ion; electrospray ionization (ESI) voltage, 1.65 KV; nebulizing gas (N2) flow rate, 1.5 l/min; and drying gas flow rate, 10.0 l/min. The desolation line was heated to 250°C and the heat block was heated to 450°C. Collision-induced dissociation gas pressure was set to 230 kPa. The MS data were acquired from m/z 100 to 2,000. The data were analyzed using Lab Solutions software (version 5.75, Shimadzu Corp.).
Data were analyzed using SPSS 20.0 statistical software (IBM Corporation) and presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and the univariate analysis of general linear model were used to determine statistical significance. Tukey's test was applied as the post hoc test. The data of western blot analysis were quantified using Image J software version 1.52a (National Institute of Health). A value of P<0.05 was considered to indicate a statistically significant difference.
The present study first investigated the inhibitory effects of AKH at multiple concentrations (0, 50, 100, 150, 200, 300 and 400 µg/ml) on the proliferation of various cancer cells and compared to normal hepatic stellate LX-2 cells by CCK-8 assay. The results revealed that AKH significantly (P<0.001) inhibited the growth of various cancer cells in a concentration-dependent manner; however, it exerted a weak inhibitory effect on the LX-2 cells (
Apoptosis is closely related to the fate of cancer and is one of the key targets for novel anticancer drug discovery and development (
For the quantitative analysis of the effects of AKH on apoptosis induction, flow cytometric analysis was then performed using Annexin-V-FITC/PI double staining assay in the Panc-28 and A549 cells. The data demonstrated that treatment with AKH for 48 h markedly induced apoptosis in a concentration-dependent manner; the apoptotic rates were 7.23±0.45 and 7.58±0.93%, 9.68±0.73 and 12.68±0.53%, 16.89±1.22 and 16.23±0.52%, and 36.45±1.65 and 32.35±.26% for the Panc-28 and A549 cells treated with the vehicle (control), or 150, 200 and 250 µg/ml of AKH, respectively (
To further investigate the underlying mechanisms of the AKH-induced apoptosis of the cancer cells, the expression of cleaved PARP, caspase-8, caspase-3 and caspase-9 was examined using western blot analysis of the Panc-28 and A549 cells. The results revealed that the relative expression levels of the apoptotic proteins, cleaved PARP, caspase-8, cleaved caspase-3 and caspase-9, were significantly increased in a concentration-dependent manner following treatment with AKH (
Autophagy plays a dual role in cancer initiation and progression, autophagy and apoptosis are highly interconnected in determining the fate of cancer cells (
To further investigate the association between AKH-induced autophagy and apoptosis, the Panc-28 and A549 cells were pre-treated with the autophagy inhibitors, 3MA (5 mM) or Baf-A1 (10 nM), for 24 h followed by treatment with AKH (250 µg/ml) for 48 h. The cells were then examined by flow cytometry and western blot analysis. The results of CCK-8 assay revealed that both the autophagy inhibitors, 3MA and Baf-A1, significantly decreased the inhibitory effects of AKH on the viability of the Panc-28 (P<0.001;
AMPK and Akt/mTOR/p70S6K play a critical role in regulating both the apoptosis and autophagy of cancer cells (
These data indicated that AKH significantly increased the levels of p-AMPK and decreased those of p-Akt, and its downstream target proteins, such as p-mTOR and p-70S6K in the Panc-28 and A549 cells.
After completing the experiments to determine the effects of AKH on the growth inhibition and apoptosis/autophagy induction of the Panc-28 and A549 cells
Finally, the present study determined the compositions of AKH using LCMS-IT-TOF assay with 0.5% formic acid in water and acetonitrile for separation. A total of nine components were detected from AKH using this assay (
Cancer is one of the most critical public health concerns and a leading cause of morbidity and mortality worldwide (
Firstly, the present study investigated the anticancer effects of AKH in seven cancer cell lines, including Panc-28, A549, MDA-MB-468, Hela, A875, U87 and HCT-116, and compared these to those in normal human hepatic stellate LX-2 cells using CCK-8 assay. The results revealed that AKH displayed potent cytotoxicity against the tested cancer cells with IC50 values of 203–284 µg/ml; the Panc-28 and A549 cells were the most sensitive cells as regards the response to AHK treatment (
Apoptosis and autophagy play a critical role in the fate of cancer and cancer therapy (
It has been demonstrated that oridonin-induced apoptosis is attenuated by 3MA (an autophagy specific inhibitor) in human breast cancer cells, indicating that oridonin-induced autophagy participated in the upregulation of apoptosis (
Akt/mTOR/p70S6K is one of the major pathways of autophagy and the activation of Akt/mTOR/p70S6K can inhibit autophagy (
To successfully develop an effective novel anticancer drug clinically, it must be validated using
Furthermore, the compositions of AKH were determined by LCMS-IT-TOF analysis and nine compounds were detected from AKH namely pinocembrin, trans,trans-1,7-diphenyl–1,3-heptadien-5-ol, cardamomin, (−)-2-(1,2,3,4,4a,7-haxahydro-4a,8-dimethyl-1,7-dioxo-2-naphthyl)-propionic acid, (1E,4Z)-5-hydroxy-1-phenylhexa-1,4-dien-3-one, trans,trans-1,7-diphenyl-5-hydroxy-4,6-hepten-3-one, 1,7-diphenyl-4,6-heptadien-3-one, katsumadain B and (4E,6E)-5-hydroxy-1,7-diphenylhepta-4,6-dien-3-on-e, respectively (
Finally, the model of the possible underlying molecular mechanisms associated with the effects of AKH on the apoptosis and autophagy in human cancer cells was proposed in (
In conclusion, the present study demonstrated that AKH selectively inhibited the proliferation of various cancer cells, whereas it exerted much less potent inhibitory effects on normal liver cells. AKH significantly induced cellular apoptosis and autophagy by regulating the AMPK and Akt/mTOR/p70S6K signaling pathways in Panc-28 pancreatic cancer and A549 lung cancer cells
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SC and XL provided the funding and designed the study, supervised the experiments, and analyzed the data. WA, YZ, HL and YZ performed the experiments. HZ, GZ, YL and ML analyzed the data and prepared the figures. WA, YZ and SC wrote the manuscript. WA and HL confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.
All animal experiments were approved (permit no. 201802-112) by the Institutional Animal Care and Use Committee of Southwest Medical University (Luzhou, China) and strictly followed the guidelines for the investigation of experimental pain in conscious animals for improving animal welfare to minimize animal suffering (
Not applicable.
The authors declare that they have no competing interests.
AKH inhibits the proliferation of various cancer cells, as demonstrated using CCK-8 assay. (A) Viability of Panc-28, A549, MDA-MB-468, Hela, A875, U87, HCT-116 and LX-2 cells following treatment with the vehicle control or various concentrations of AKH (50, 100, 150, 200, 300 and 400 µg/ml) for 48 h. (B) Time-effects of AKH on (a) Panc-28 and (b) A549 cells. The cells were treated with the vehicle control or AKH at 100, 150, 200, 250, 300 and 400 µg/ml for 24, 48, or 72 h, respectively. The data are representative of three independent experiments (n=3) run in triplicate and are expressed as the mean ± SD. ***P<0.001 vs. LX-2 cells in A and vs. 24 h in B (determined using one-way ANOVA). AKH,
Effects of AKH on apoptosis and the expression of apoptosis-related proteins in Panc-28 and A549 cells. (A) The morphological features of apoptosis induced by AKH in Panc-28 by Hoechst 33342/PI staining assay. (B) The morphological features of apoptosis induced by AKH in A549 cells by Hoechst 33342/PI staining assay. (C) Analysis of apoptosis of Panc-28 cells examined by flow cytometry with Annexin-V-FITC/PI double staining assay. (D) Analysis of apoptosis of A549 cells by flow cytometry with Annexin-V-FITC/PI double staining assay. (E) Percentage of apoptotic Panc-28 cells. (F) Percentage of apoptotic A549 cells. (G) The expression of apoptosis-related proteins PARP, cleaved-PARP, caspase-8, pro-caspase-3, cleaved-caspase-3 and caspase-9 in Panc-28 cells by western blot analysis. (H) The expression of apoptosis-related proteins PARP, cleaved-PARP, caspase-8, pro-caspase-3, cleaved-caspase-3 and caspase-9 in A549 cells by western blot analysis. (I) The protein expression of cleaved-PARP/PARP, caspase-8, cleaved-caspase-3/pro-caspase 3 and caspase-9 in Panc-28 cells. (J) The protein expression of cleaved-PARP/PARP, caspase-8, cleaved-caspase-3/pro-caspase-3 and caspase-9 in A549 cells. Actin was used as a loading control. The cells were treated with the vehicle control or AKH at 150, 200 and 250 µg/ml for 48 h. The data are representative of three independent experiments (n=3) run in triplicate and are expressed as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 vs. vehicle control (determined using one-way ANOVA). AKH,
Effects of the autophagy inhibitors, 3MA and Baf-A1, on AKH-induced cell growth inhibition, apoptosis, and the expression of apoptosis- and autophagy-related proteins in Panc-28 and A549 cells. (A) The viability of the Panc-28 cells by CCK-8 assay. (B) The viability of the A549 cells by CCK-8 assay. (C) Flow cytometric analysis of Panc-28 cells. (D) Flow cytometric analysis of A549 cells. (E) The bands of apoptosis-related proteins PARP, cleaved-PARP, pro-caspase-3 and cleaved-caspase-3; autophagy-related proteins LC3, Beclin-1 and actin following treatment of 3MA and AKH alone or in combination in Panc-28 and A549 cells by western blot analysis. (F) The bands of apoptosis-related proteins PARP, cleaved-PARP, pro-caspase-3 and cleaved-caspase-3, autophagy-related proteins LC3, Beclin-1 and actin following treatment of Baf-A1 and AKH alone or in combination in Panc-28 and A549 cells by western blot analysis. Actin was used as a loading control. The cells were pre-treated with the vehicle control, 3MA (5 mM) or Baf-A1 (10 nM) for 24 h followed by the vehicle control or AKH (250 µg/ml) for 48 h. The data are representative of three independent experiments (n=3) run in triplicate and are expressed as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 vs. vehicle control (determined using one-way ANOVA). AKH,
The effect of AKH on the expression of proteins related to the AMPK and Akt/mTOR/p70S6K singling pathways in Panc-28 and A549 cells by western blot analysis. (A) Bands of Akt, pAkt, AMPK, pAMPK, mTOR, pmTOR, 70S6K p70S6K and Actin in Panc-28 cells. (B) Bands of Akt, pAkt, AMPK, pAMPK, mTOR, pmTOR, 70S6K p70S6K and Actin in A549 cells. Actin was used as a loading control. (C) Relative protein expression of pAkt/Akt, pAMPK/AMPK, pmTOR/mTOR and p70S6K/70S6K in Panc-28 cells. (D) Relative protein expression of pAkt/Akt, pAMPK/AMPK, pmTOR/mTOR and p-p70S6K/p70S6K in A549 cells. The cells were treated with the vehicle control or AKH at 150, 200, and 250 µg/ml for 48 h. The data are representative of three independent experiments (n=3) run in triplicate and are expressed as the mean ± SD. **P<0.01 and ***P<0.001 vs. vehicle control (determined using one-way ANOVA). AKH,
AKH inhibits the tumor growth of A549 ×enografts in nude mice
Analysis of the composition from AKH by LCMS-IT-TOF. (A) The BPC spectrum of AKH. (B) Chemical structures of AKH compounds detected by LCMS-IT-TOF. The nine compounds (
A proposed schematic model of the underlying molecular mechanism associated with the effects of AKH on apoptosis and autophagy in human cancer cells. The black lines indicate activation and the green lines indicate inhibition. AKH,
IC50 values of AKH in various cell lines determined by CCK-8 assay.
Cell line | Cell type | IC50 (µg/ml) |
---|---|---|
Panc-28 | Human pancreatic cancer | 202.7±6.4 |
A549 | Human lung cancer | 219.0±9.9 |
MDA-MB-468 | Human breast cancer | 234.7±8.4 |
Hela | Human cervical cancer | 236.2±8.8 |
A875 | Human melanoma cancer | 259.6±11.3 |
U87 | Human glioblastoma of unknown origin | 262.0±15.9 |
HCT-116 | Human colon cancer | 284.0±9.1 |
LX-2 | Human hepatic stellate cell | 395.4±2.21 |
Analysis of the characterized compounds from AKH.
Compound no. | Retention time (min) | Molecular weight (Da) | Molecular formula | Unsaturation | MS fragmentation (error in mDa) | Ultraviolet absorption (λmax) nm | Structure or type of compound |
---|---|---|---|---|---|---|---|
1 | 7.78 | 256 | C15H12O4 | 10 | Pos: 257.0889 [(M+H)+, 8.1] | 216, 289 | Pinocembrin |
Neg: 255.0680 [(M-H)−, 1.7] | |||||||
2 | 9.33 | 264 | C19H20O | 10 | Pos: 265.1605 [(M+H)+, 1.8] | 250, 331 | Trans,trans-1,7-diphenyl-1,3-heptadien-5-ol |
MS/MS 247.1500 [(M+H)+, 1.9] | |||||||
3 | 10.28 | 270 | C16H14O4 | 10 | Pos: 271.0995 [(M+H)+,3.0] | 215, 344 | Cardamomin |
Neg:269.0787 [(M-H)-, −3.2] | |||||||
4 | 10.92 | 262 | C15H18O4 | 7 | Pos: 263.1350 [(M+H)+, 7.2] | 218, 250 | (−)-2-(1,2,3,4,4a,7-Hexahydro-4a,8-dimethyl-1,7-dioxo-2-naphthyl)-propionic acid |
5 | 11.26 | 188 | C12H12O2 | 7 | Pos: 189.0877 [(M+H)+, 3.3] | 225, 340 | (1E,4Z)-5-Hydroxy-1-phenylhexa-1,4-dien-3-one |
6 | 12.17 | 278 | C19H18O2 | 11 | Pos: 279.1347 [(M+H)+, 2.0] | 219, 331 | Trans,trans-1,7-diphenyl-5-hydroxy-4,6-hepten-3-one |
7 | 16.65 | 262 | C19H18O | 11 | Pos: 263.1498 [(M+H)+, 6.8] | 231, 355 | 1,7-Diphenyl-4,6-heptadien-3-one |
8 | 18.18 | 476 | C32H28O4 | 19 | Pos: 477.2004 [(M+H)+, −5.6] | 229, 325 | Katsumadain B |
Neg:475.1913 [(M-H)−, −0.2] | |||||||
9 | 19.51 | 278 | C19H18O2 | 11 | Pos: 279.1346 [(M+H)+, 2.0] | 229, 361 | (4E,6E)-5-Hydroxy-1,7-diphenylhepta-4,6-dien-3-one |
Neg: 277.1174 [(M-H)−, −6.0] |
Pos, detection in positive ion mode; Neg, detection in negative ion mode.