Akebia Fructus has long been used for hepatocellular carcinoma (HCC) in China, while the molecular mechanism remains obscure. Our recent work found that
According to the Word Cancer Report from IARC (International Agency for Research on Cancer), human hepatocellular carcinoma (HCC) is the sixth most common malignant cancer throughout the world and the third in China; the cases in China are more than half of the total worldwide [
Akebia Fructus is the near-mature dried fruit of plant
Akebia Fructus is always used as a whole fruit, which is composed of the pulp and the seed. In our recent research, extracts of different parts of
Ethanol, Petroleum ether, ethyl acetate, and n-butanol are all AR grade and purchased from Sinophar Chemical Reagent Co., Ltd. (Shanghai, China). Trizol, G418, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Life Technologies (Carlsbad, CA, USA). Rabbit anti-SEC63 (1 : 800) polyclonal and rabbit anti-DNAJB11 (1 : 500) polyclonal were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-HSP90AA1 (1 : 1000) was purchased from Stressgen (Enzo Life Sciences, NY, USA). Rabbit anti-monoclonal HYOU1 (1 : 5000) was purchased from Epitomics (Abcam, CA, USA). Mouse monoclonal anti-GRP78 (1 : 1000), mouse monoclonal anti-HSPA9 (1 : 1000), and mouse monoclonal anti-GAPDH (1 : 2500) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary antibodies HRP Goat anti-mouse IgG and HRP Donkey anti-rabbit IgG were purchased from Biolegend (San Diego, CA, USA).
The whole fruit of
The seed was peeled off from the whole fruit and dried at 60°C for 24 hours; dried seed (800 g) was smashed and soaked in 75% ethanol for 2 hours at room temperature and then extracted by 8 L 75% ethanol reflux at 80°C for 2 hours and filtered by gauze; the filtration residue was extracted again under the same condition; all resulting filtrations were combined and concentrated by Rotavapor (BÜCHI Labortechnik, Switzerland) under reduced pressure; 2700 mL concentrated extract was obtained, followed by successive extraction with the same volume of Petroleum ether, ethyl acetate, and water-saturated n-butanol, three times in each solvent; this procedure resulted in three extracts; the n-butanol soluble extract was further concentrated by Rotary Evaporator (IKA, Germany) at 60°C (20–40 rpm); 310 mL extractum was obtained and then freeze-dried to 33.26 g power, which is simply called ATSE in experiment. The extract yield was 4.26% (w/w). ATSE was diluted to 0.5 g/mL by distilled water and then dissolved in the RPMI 1640 culture medium to 10 mg/mL and finally filtered through a 0.45
HepG2, HuH7, and SMMC-7721 cell lines were obtained from Chinese Academy of Sciences (Shanghai, China), cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100
Cells were split and seeded in 96-well and 6-well plates, 18–24 hours after seeding, and when the confluency was about 70%, G418 or ATSE was added to four different groups according to the requirement: untreated control; G418 group (0.45 mg/mL); ATSE group (0.625 mg/mL); the combination group was composed of half dose of each (0.225 mg/mL G418 plus 0.31 mg/mL ATSE). 72 hours after treatment, cells in 96-well plates were used for MTT assay, and the cell morphology of 6-well plates was firstly examined under an inverted phase contrast microscope (Olympus CKX41, Tokyo, Japan), then for the other different assays.
Cell survival was assessed using an MTT assay at 72 hours after treatment; the culture medium of 96-well plate was removed and replaced with 0.5 mg/mL MTT solution. After 4 h incubation at 37°C, this solution was removed and the resulting blue formazan was solubilized in 100
Treatments of cells were performed according to the protocol of the PI detection kit (KeyGEN Biotech, Shanghai, China). Briefly, cells were harvested and resuspended in cold PBS at a concentration of
Cell lysates were harvested in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS). The whole cell lysates were centrifuged at 12,000 rpm for 15 minutes at 4°C; then the supernatant was collected and protein concentration was measured by Pierce BCA Protein Assay Kit (Thermo scientific, UT, USA). Cell lysates (20
Total RNA was isolated from cells with Trizol according to manufacturer’s protocol. The qualities of all RNA samples were monitored: the absorbance at 260 nm and 280 nm was measured by NanoDrop 1000 (Thermo Fisher Scientific, MA, USA) and acceptable A260/280 ratios were in the range of 1.7–2.1; the RNA 6000 Nano kit (Agilent Technologies, CA, USA) was used to detect RIN (RNA integrity number) and 28S/18S ratios by Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA); the former was approximately 9.2 and the latter were approaching 2 : 1. High-quality total RNA was used as a starting material for making total RNA/Poly-A RNA controls and was mixed using a GeneChipR Poly-A RNA Control Kit (Affymetrix, CA, USA). Then cDNA was synthesized using an Ambion WT Expression Kit (Affymetrix, CA, USA), followed by ssDNA fragmentation and labeling of fragmented ssDNA with GeneChip WT Terminal Labeling Kit (Affymetrix, CA, USA). The biotin-labeled fragmentations were hybridized to a gene chip (Affymetrix HuGene 1.1 Sense Target Array) at 48°C for 16 h. Following hybridization, the chips were washed and stained in the GeneAtlas Fludic Station (Affymetrix, CA, USA). Then the arrays were put into the GeneAtlas Imaging Station (Affymetrix, CA, USA). Microarray data were analyzed by Affymetrix Expression Console (Affymetrix, CA, USA). The data were normalized using the iterative PLIER default protocol. Changes in gene expressions were analyzed and compared with untreated control and the criteria for positive fold change are greater than 1.5-fold increase or decrease.
Data were analyzed using the IBM statistics SPSS 19.0 software. The results are presented as means ± standard deviation (S.D.) and the comparison between groups was analyzed by one-way analysis of variance (ANOVA). A value of
The morphological changes induced by ATSE were observed. HepG2 cells were not sensitive to 0.45 mg/mL G418 (Figure
ATSE cause different morphological changes in HCC cell lines. G418 (0.45 mg/mL), ATSE:
In brief, ATSE induced ER stress in these three HCC cell lines but the degree varied and it seemed that SMMC-7721 cells were the most sensitive kind.
To assess the effect of ATSE on cell viability, MTT assay was performed. As shown in Figure
ATSE suppress cell viability in HCC cell lines. Cell viability was tested by MTT. G418 (0.45 mg/mL), ATSE:
To figure out whether the proliferation inhibition is due to cell cycle arrest, the cell cycle analysis by Flow cytometry was performed, and these HCC cell lines showed different features as shown below.
Firstly, In HepG2 cells, at 72 hours after G418 incubation, the percentage of cells in G0/G1 phase decreased significantly to 52.9% versus 59.7% of the control (Figures
Different cell cycle influences of ATSE on HCC cell lines. Cell cycle analysis was performed by Flow Cytometry. G418 (0.45 mg/mL); ATSE:
In HuH7 cells (Figures
Unlike HepG2 or HuH7, basically ATSE does not influence much cell cycles of SMMC-7721 (Figures
To further clarify the molecular mechanism of ATSE induced ER stress in HepG2, HuH7, and SMMC-7721 cells, some ER stress-related proteins were chosen and examined by Western blot analysis. As indicated in Figure
Effect of ATSE on the protein expression of ER stress-related biomarkers. The relative expression of ER-stress biomarkers at protein level after 72-hour treatment. GAPDH was used as internal control. G418 (0.45 mg/mL); ATSE:
For full characterization of the molecular mechanism of ATSE, Affymetrix array was used. 82 ER stress-related genes were selected one by one manually from NCBI gene database. There were significant changes in the levels of gene expression in 8 of 82 genes (Table
List of ER stress-related genes with significant expression in HepG2 cells (fold change ≥1.5-fold).
Probe set ID | Gene symbol | Control | G418 | ATSE | 1/2(G418 + ATSE) | ATSE/control |
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8152628 | DERL1 | 199 | 336 | 355 | 694 | 1.78 |
7954196 | MGST1 | 733 | 958 | 1305 | 1663 | 1.78 |
7995895 | HERPUD1 | 714 | 1239 | 1166 | 2351 | 1.63 |
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7956593 | OS9 | 315 | 357 | 475 | 407 | 1.51 |
8156838 | SEC61B | 528 | 426 | 348 | 631 | −1.52 |
8080084 | MANF | 495 | 587 | 320 | 791 | −1.55 |
8075182 | XBP1 | 758 | 583 | 352 | 343 | −2.16 |
G418 (0.45 mg/mL); ATSE:
There were significant changes in the levels of gene expression in 38 of 82 genes (Table
List of ER stress-related genes with significant expression in HuH7 cells (fold change ≥1.5-fold).
Probe set ID | Gene symbol | Control | G418 | ATSE | 1/2(G418 + ATSE) | ATSE/control |
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7954196 | MGST1 | 479 | 815 | 1026 | 720 | 2.14 |
8024754 | CREB3L3 | 171 | 55 | 353 | 69 | 2.06 |
7967563 | UBC | 2502 | 4281 | 4550 | 7132 | 1.82 |
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7949383 | SYVN1 | 326 | 235 | 208 | 224 | −1.57 |
7964460 | DDIT3 | 239 | 137 | 151 | 332 | −1.58 |
8125295 | ATF6B | 202 | 49 | 117 | 82 | −1.73 |
8080084 | MANF | 268 | 490 | 152 | 351 | −1.77 |
8075182 | XBP1 | 820 | 705 | 448 | 596 | −1.83 |
8091458 | SERP1 | 561 | 1050 | 303 | 635 | −1.85 |
8137526 | INSIG1 | 214 | 356 | 114 | 290 | −1.88 |
7916432 | DHCR24 | 2595 | 560 | 1278 | 280 | −2.03 |
8138108 | KDELR2 | 1272 | 1329 | 622 | 605 | −2.04 |
8160914 | VCP | 423 | 764 | 200 | 227 | −2.11 |
7989619 | PPIB | 1939 | 2258 | 917 | 2377 | −2.11 |
8066889 | STAU1 | 398 | 248 | 187 | 98 | −2.12 |
8128111 | UBE2J1 | 474 | 344 | 199 | 183 | −2.38 |
7916120 | TXNDC12 | 817 | 404 | 330 | 172 | −2.48 |
7958644 | ATP2A2 | 492 | 570 | 186 | 206 | −2.65 |
8026106 | CALR | 1329 | 1694 | 497 | 1060 | −2.67 |
8051998 | MCFD2 | 411 | 507 | 149 | 213 | −2.76 |
7995895 | HERPUD1 | 915 | 870 | 297 | 960 | −3.08 |
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7906819 | ATF6 | 268 | 361 | 80 | 174 | −3.33 |
8041967 | ERLEC1 | 275 | 363 | 77 | 109 | −3.57 |
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8135480 | DNAJB9 | 256 | 431 | 53 | 351 | −4.86 |
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7956593 | OS9 | 1008 | 857 | 195 | 383 | −5.17 |
8046759 | DNAJC10 | 452 | 202 | 77 | 27 | −5.87 |
8023561 | LMAN1 | 941 | 1298 | 159 | 448 | −5.93 |
7980547 | SEL1L | 333 | 295 | 46 | 68 | −7.27 |
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7904881 | PDIA3P | 1192 | 1261 | 131 | 644 | −9.11 |
7958130 | HSP90B1 | 1813 | 2793 | 198 | 1396 | −9.17 |
7983274 | PDIA3 | 1181 | 1226 | 119 | 602 | −9.95 |
8095628 | ALB | 3131 | 1860 | 63 | 435 | −49.59 |
G418 (0.45 mg/mL); ATSE:
There were significant changes in the levels of gene expression in 19 of 82 genes (Table
List of ER stress-related genes with significant expression in SMMC-7721 cells (fold change ≥1.5-fold).
Probe set ID | Gene symbol | Control | G418 | ATSE | 1/2(G418 + ATSE) | ATSE/control |
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7964460 | DDIT3 | 106 | 274 | 525 | 546 | 4.94 |
8076481 | CYB5R3 | 262 | 209 | 539 | 279 | 2.06 |
8139003 | HERPUD2 | 186 | 263 | 323 | 271 | 1.74 |
8042107 | VRK2 | 536 | 973 | 884 | 1075 | 1.65 |
8156838 | SEC61B | 299 | 566 | 469 | 638 | 1.57 |
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8062174 | ERGIC3 | 984 | 1042 | 662 | 686 | −1.50 |
7958644 | ATP2A2 | 575 | 769 | 382 | 536 | −1.50 |
7989619 | PPIB | 1595 | 2090 | 989 | 2036 | −1.61 |
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8178498 | HLA-B | 327 | 268 | 190 | 246 | −1.72 |
7958130 | HSP90B1 | 1426 | 2138 | 744 | 2022 | −1.92 |
8091954 | GOLIM4 | 641 | 378 | 329 | 364 | −1.95 |
7983274 | PDIA3 | 855 | 1271 | 437 | 949 | −1.96 |
8023561 | LMAN1 | 735 | 816 | 372 | 690 | −1.98 |
7904881 | PDIA3P | 889 | 1360 | 440 | 980 | −2.02 |
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8160914 | VCP | 333 | 318 | 114 | 278 | −2.91 |
G418 (0.45 mg/mL); ATSE:
Affymetrix array data also presented genes expressions information of KEGG integrin-mediated cell adhesion pathway, which helps to explain the effect of ATSE on HepG2 cells. There were significant changes in 6 of 89 genes, 4 were upregulated and 2 were downregulated (Table
List of genes with significant expression in the KEGG integrin-mediated cell adhesion pathway for HepG2 cells (fold change ≥1.5-fold).
Probe set ID | Gene symbol | Control | G418 | ATSE | 1/2(G418 + ATSE) | ATSE/control |
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7983763 | MAPK6 | 233 | 314 | 382 | 405 | 1.64 |
8046380 | ITGA6 | 216 | 323 | 385 | 220 | 1.78 |
8051670 | SOS1 | 291 | 386 | 520 | 515 | 1.79 |
8084963 | PAK2 | 173 | 252 | 360 | 338 | 2.08 |
8090162 | ITGB5 | 330 | 357 | 220 | 150 | −1.50 |
8111915 | SEPP1 | 3337 | 2847 | 1761 | 1131 | −1.87 |
G418 (0.45 mg/mL); ATSE:
89 genes in the KEGG apoptosis pathway were displayed in Affymetrix array data. There were significant changes in the levels of gene expression in 6 of 89 genes, 5 were upregulated and 1 was downregulated (Table
List of genes with significant expression in the KEGG-apoptosis pathway for HuH7 cells (fold change ≥1.5-fold).
Probe set ID | Gene symbol | Control | G418 | ATSE | 1/2(G418 + ATSE) | ATSE/control |
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8149733 | TNFRSF10B | 408 | 688 | 794 | 805 | 1.95 |
7966746 | HRK | 153 | 188 | 252 | 403 | 1.65 |
8065569 | BCL2L1 | 1172 | 1558 | 1822 | 1293 | 1.55 |
8012257 | TP53 | 212 | 209 | 320 | 66 | 1.51 |
7956989 | MDM2 | 766 | 1242 | 1138 | 680 | 1.50 |
7924733 | PARP1 | 102 | 90 | 67 | 56 | −1.50 |
G418 (0.45 mg/mL); ATSE:
Akebia Fructus has been long used for HCC in TCM in China, in the manner of one member in herbal formula under most circumstances. The data from clinical epidemiological investigation on 2060 HCC cases [
Therefore, in our recently work [
Our data exhibited that ATSE leads to different degrees of ER stress in these HCC cell lines and a decrease in cell viability (Figures
As shown in Figure
Besides morphological changes and cell cycle alterations, ER stress is often accompanied with expression changes of a lot of genes and proteins, or called as biomarkers. Generally, this event started from three transmembrane sensors in UPR (unfolded protein response) pathway [
Furthermore, even Affeymetrix array presented such a comprehensive gene expression profile; how ER stress was induced and how these genes or proteins are influenced by ATSE, active or passive, directly or indirectly remain unclear. Therefore, besides the continued analysis of these data, further investigations, such as cotreatment with some mechanism-known ER-stress-induced agents and such as genes knocked down by RNA interference, are beneficial to know more about how ATSE works and the role of those significantly changed genes or proteins.
The phytochemistry of ATSE is not discussed in the present work, for the main ingredients are almost clear, which are triterpenoid saponins. Since the work of Ryuichi et al. in the last century [
The effect of drug combination is also worthy of attention. As shown in the MTT result (Figure
In summary, our data demonstrated that the
Human hepatocellular carcinoma
Traditional Chinese medicine
3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
Endoplasmic reticulum
Kyoto Encyclopedia of Genes and Genomes.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Wen-Li Lu and Hong-Yan Ren contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (no. 30973703 and no. 81273641).