Hepatoma is a common malignant tumor. Thus, the development of a high-efficacy therapeutic drug for hepatoma is required. In this study, (+)-terrein isolated from the marine sponge-derived
Hepatocellular carcinoma (HCC) is the most common malignant tumor and the third leading cause of cancer mortality worldwide (
In antitumor therapy, numerous drugs affect tumorigenesis and tumor growth; however, the key is determining which drugs to exploit in the areas of signal transduction, cell-cycle regulation, apoptosis, telomere biology, necrosis, autophagy, cell senescence and angiogenesis (
Findings of studies showed that the isolation and production of (+)-terrein (the molecular formula is C8H10O3 and the molecular weight is 154;
(+)-Terrein (
An MTT assay [3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was performed with ~4×103 cells/well in 96-well plates. The plates containing the cells were incubated at 37°C for 24 h, and the cells were treated with (+)-terrein at 37°C for 48 h. The protocol was performed using an MTT cell proliferation and cytotoxicity detection kit (KeyGen, Nanjing, China) according to the manufacturer’s instructions. Briefly, DMEM was supplemented with 50 μl of MTT reagent to each well and incubated at 37°C for 4 h. Thereafter, the MTT solution was removed. Following the addition of 150 μl of dimethyl sulfoxide (DMSO), the plates were incubated at 37°C for 15 min to dissolve the formazan crystals. Absorbance of DMSO extracts was detected at 550 nm by using an Enspire 2300 microplate reader (PerkinElmer, Foster City, CA, USA). A total of ~3×105 cells/well were inoculated in 6-well plates at 37°C for 24 h and treated with (+)-terrein at 37°C for 48 h. The PBS-treated cells served as controls. These cells were used to detect cell morphology, cell cycle, apoptosis, and RNA extraction.
Cells (3×105) were cultured in 6-well plates at 37°C for 24 h. The Bel-7402 and A549 cells were then treated with 10 μM and 10 mM (+)-terrein at 37°C for 48 h, respectively, and the PBS-treated cells served as controls. The treated cells were used to observe cell morphology and apoptosis. Light microscopy images of the cells were captured using a Nikon Eclipse Ti-inverted microscope and a Nikon digital sight s-Qi1Mc camera (both from Yokohama, Japan). For each surface, three non-overlapping images were selected.
The cells were rinsed once in chilled PBS, digested with 0.25% trypsin (Gibco), and then resuspended in DMEM and 10% serum. The suspended cells were centrifuged at 2,000 × g at 4°C for 5 min and washed once in cold PBS. The cells were stained with Alexa Fluor® 488 Annexin V and PI by using an Alexa Fluor® 488 Annexin V/Dead cell apoptosis kit (Invitrogen, New York, USA) according to the manufacturer’s instructions. The stained cells were analyzed using flow cytometry (FACSAria-II, BD Biosciences, San Jose, CA, USA).
The Bel-7402 cells treated with 10 μM (+)-terrein were trypsinized and washed with a PBS buffer. Fifty microliters of single cell suspension was examined using a cell cycle detection kit (KeyGen). The stained cells were analyzed using flow cytometry (FACSAria II).
The Bel-7402 cells treated with (+)-terrein or PBS were trypsinized and washed with a PBS buffer. The cells were collected using centrifugation at 2,000 × g for 5 min. The cell pellet was then resuspended in RL buffer and centrifuged at 13,000 × g for 5 min. Total RNA was extracted according to the manufacturer’s instructions (CWBio, Beijing, China). The purity and concentration of RNA was confirmed by the relative absorbance ratio at 260/280 nm and 260 nm, respectively, by using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
Reverse transcription was performed according to the manufacturer’s instructions (Thermo Fisher Scientific). Total RNA (1 μg) was mixed with 4 μl of a 5X reaction buffer, 1 μl of oligo (dT)18 primer, 1 μl of RiboLock RNase inhibitor, 2 μl of a 10 mM dNTP mix, 1 μl of RevertAid reverse transcriptase (100 U/μl), and then, ddH2O was added to increase the volume to 20 μl. Reverse transcription was performed at 37°C for 60 min and then at 70°C for 5 min. The resulting cDNA was stored at −70°C until use.
Each cDNA was diluted to 1 ml with ddH2O and mixed with 1 ml of 2X SYBR Premix Ex Taq™ (Takara, Dalian, China). Twenty microliters of this mixture was added to each well of a 96-well PCR array, except for genomic DNA control (GDC). The 96-well PCR plates containing gene primers were prepared by the CT Bioscience Company (Changzhou, China). The sealed PCR plate was loaded in an Eppendorf Realplex 4S (Hamburg, German). The PCR was performed under the following conditions: 95°C for 5 min, 40 cycles at 95°C for 15 sec, 60°C for 15 sec, and 72°C for 20 sec. The melting curve procedure (95°C for 15 sec, 60°C for 15 sec, and 95°C for 15 sec) was implemented to analyze the PCR specificity. Dissociation curves (DC) and melting temperatures (Tm) were recorded. Relative changes in gene expression were calculated using the threshold cycle (Ct) method (
Statistical product and service solutions (SPSS ver. 13.0) was used for the data analysis. All of the experiments were conducted in duplicate. The results are presented as mean ± SD (standard deviation) unless otherwise specified. The P-values were two-tailed. P≤0.05 was considered to indicate a statistically significant difference. The cell-cycle pathway is a highly regulated process that incorporates three major checkpoints including the participation of several genes (
The
We observed a marked phenomenon regarding cell morphology alterations when cells were treated with (+)-terrein. Morphological changes in the Bel-7402 cells exposed to 10 μM (+)-terrein and in the A549 cells exposed to 10 mM (+)-terrein for 48 h were examined using light microscopy (
The cell morphology gene (FN, N-cadherin, and vimentin) expression of the Bel-7402 cells treated with 10 μM (+)-terrein was investigated. The results showed that the gene expression of FN, N-cadherin, and vimentin decreased −3.61-, −2.39-, and −2.05-fold, respectively, compared with the control (
To determine whether this reduction in cell growth induced by (+)-terrein was mediated by apoptosis, flow cytometric analysis was performed using PI and Annexin-V staining. The results indicated that the apoptotic levels of Bel-7402 and A549 cells treated with (+)-terrein (10 μM and 10 mM) did not increase (
Cell proliferation depends on the specific progression of the cell cycle (
In this study, high-throughput gene expression analysis of 73 genes was performed (
Numerous drugs affect tumorigenesis and tumor growth through several mechanisms, including signal transduction, cell-cycle regulation, apoptosis, telomere biology, angiogenesis and cell senescence (
Since the adhesion and distribution of anchorage-dependent cells are prerequisites for cell viability and proliferation, cell growth and survival also depend on cell morphology (
The inhibitory mechanism of (+)-terrein against the Bel-7402 cell differed from the apoptosis of breast cancer and pulmonary tumor cell lines (
Kim
Cell-cycle progression in mammalian cells is regulated by various proteins (
Eight genes are related to the cell cycle (
Membrane-associated tyrosine- and threonine-specific CDC2-inhibitory kinase encoded by the
In conclusion, (+)-terrein exhibited cytotoxicity against the Bel-7402 human hepatoma cell line, yielding an IC50 value of 11.63 μM ±0.02. In addition, (+)-terrein induced round-cell morphology of Bel-7402 and A549 cells but did not induce cell apoptosis. Furthermore, (+)-terrein inhibited Bel-7402 human hepatoma cell proliferation and arrested the cell cycle in the G2/M phase by breaking down the expression of the
We would like to thank Dr Qian Luo at the Instrument Sharing and Technical Service Platform of SJTU for technical advice on using flow cytometry. We would also like to thank Dr Valliappan Karuppiah for providing assistance with the language. This study was supported by the High-Tech Research and Development Program of China (2011AA090702), the Medical and Engineering Cross Funds of Shanghai Jiao Tong University (YG2011ms13), and the National Natural Science Foundation of China (J1210047 and 31300104).
anaphase-promoting complex subunit 5
cyclin/cyclin-dependent kinase
Cdk inhibitors
cyclin-dependent kinase inhibitor 1C
threshold cycle
dissociation curves
genomic DNA control
hepatocellular carcinoma
cyclin-dependent kinase 4 ihibitor B
mitotic cyclin-dependent kinase
proliferating cell nuclear antigen
Statistical Product and Service Solutions
melting temperature
Kyoto Encyclopedia of Genes and Genomes
The chemical structure of (+)-terrein.
Effect of (+)-terrein on cell viability and proliferation. (A) Effect of (+)-terrein on cell viability and proliferation of Bel-7402 cells. (B) Effect of (+)-terrein on cell viability and proliferation of A549 cells. Bel-7402 and A549 cells were treated with (+)-terrein at various concentrations for 48 h. Viability and proliferation of the cells were examined using the MTT method, and the cell survival rate (%) was calculated. Average values derived from three independent experiments shown as mean ± standard deviations. *Significant difference from control (P<0.05). **Significant difference from control (P<0.01).
Effect of (+)-terrein on cell morphology of Bel-7402 and A549 cells. Bel-7402 and A549 cells were treated with 10 μM and 10 mM (+)-terrein for 48 h, respectively. Bel-7402 and A549 cells not treated with (+)-terrein served as controls. Bars under each panel represent 50 μm.
(+)-Terrein induced cell apoptosis and necrosis. Bel-7402 and A549 cells were treated with 10 μM and 10 mM (+)-terrein for 48 h, respectively. Apoptosis and necrosis were monitored using Annexin V/PI analysis. Viable cells were Annexin V/PI-negative (Q3 quadrant). Early apoptotic cells were Annexin V-positive and PI-negative (Q4 quadrant). Late apoptotic and necrotic cells were in the Q1 and Q2 quadrants, respectively. Each bar is the mean ± SD of five independent experiments.
Graphviz view of a cell cycle canonical signaling pathway with gene data. Each box is one gene and the green boxes show the downregulated gene expression of mRNA after the Bel-7402 cells were treated with 10 μM (+)-terrein (>2-fold).
Gene information detected by the primers.
Gene ID as in PCR array | Symbol | Gene ID | Gene name |
---|---|---|---|
1 | ANAPC2 | 29882 | Anaphase promoting complex subunit 2 |
2 | ANAPC4 | 29945 | Anaphase promoting complex subunit 4 |
3 | ANAPC5 | 51433 | Anaphase promoting complex subunit 5 |
4 | BUB1 | 699 | Budding uninhibited by benzimidazoles 1 homolog (yeast) |
5 | BUB1B | 701 | Budding uninhibited by benzimidazoles 1 homolog β (yeast) |
6 | BUB3 | 9184 | Budding uninhibited by benzimidazoles 3 homolog (yeast) |
7 | CCNE1 | 898 | Cyclin E1 |
8 | CCNE2 | 9134 | Cyclin E2 |
9 | CCND1 | 595 | Cyclin D1 |
10 | CCND2 | 894 | Cyclin D2 |
11 | CCND3 | 896 | Cyclin D3 |
12 | CCNH | 902 | Cyclin H |
13 | CDC16 | 8881 | Cell division cycle 16 homolog ( |
14 | CDC20 | 991 | Cell division cycle 20 homolog ( |
15 | CDC23 | 8697 | Cell division cycle 23 homolog ( |
16 | CDC25A | 993 | Cell division cycle 25 homolog A ( |
17 | CDC25B | 994 | Cell division cycle 25 homolog B ( |
18 | CDC25C | 995 | Cell division cycle 25 homolog C ( |
19 | CDC26 | 246184 | Cell division cycle 26 homolog ( |
20 | CDC27 | 996 | Cell division cycle 27 homolog ( |
21 | CDC6 | 990 | Cell division cycle 6 homolog ( |
22 | CDC7 | 8317 | Cell division cycle 7 homolog ( |
23 | CDK4 | 1019 | Cyclin-dependent kinase 4 |
24 | CDK6 | 1021 | Cyclin-dependent kinase 6 |
25 | CDK7 | 1022 | Cyclin-dependent kinase 7 |
26 | CDKN1B | 1027 | Cyclin-dependent kinase inhibitor 1B (p27, Kip1) |
27 | CHEK1 | 1111 | Checkpoint kinase 1 |
28 | CHEK2 | 11200 | Checkpoint kinase 2 |
29 | E2F1 | 1869 | E2F transcription factor 1 |
30 | E2F2 | 1870 | E2F transcription factor 2 |
31 | E2F3 | 1871 | E2F transcription factor 3 |
32 | HDAC1 | 3065 | Histone deacetylase 1 |
33 | MAD2L1 | 4085 | MAD2 mitotic arrest deficient-like 1 (yeast) |
34 | MAX | 4149 | MYC-associated factor X |
35 | MCM2 | 4171 | Minichromosome maintenance complex component 2 |
36 | MCM3 | 4172 | Minichromosome maintenance complex component 3 |
37 | MCM4 | 4173 | Minichromosome maintenance complex component 4 |
38 | MCM5 | 4174 | Minichromosome maintenance complex component 5 |
39 | MCM6 | 4175 | Minichromosome maintenance complex component 6 |
40 | MCM7 | 4176 | Minichromosome maintenance complex component 7 |
41 | ORC1L | 4998 | Origin recognition complex, subunit 1 |
42 | ORC2L | 4999 | Origin recognition complex, subunit 2 |
43 | ORC6L | 23594 | Origin recognition complex, subunit 6 |
44 | PCNA | 5111 | Proliferating cell nuclear antigen |
45 | PKMYT1 | 9088 | Protein kinase, membrane-associated tyrosine/threonine 1 |
46 | RB1 | 5925 | Retinoblastoma 1 |
47 | RBL1 | 5933 | Retinoblastoma-like 1 (p107) |
48 | SKP2 | 6502 | S-phase kinase-associated protein 2, E3 ubiquitin protein ligase |
49 | SMC1A | 8243 | Structural maintenance of chromosomes 1A |
50 | TOP2A | 7153 | Topoisomerase (DNA) II α 170 kDa |
51 | TP53 | 7157 | Tumor protein p53 |
52 | TFDP1 | 7027 | Transcription factor Dp-1 |
53 | WEE1 | 7465 | WEE1 homolog ( |
54 | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation | ||
YWHAE | 7531 | Protein, ɛ polypeptide | |
55 | CDKN2A | 1029 | Cyclin-dependent kinase inhibitor 2A |
56 | CDKN2B | 1030 | Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) |
57 | PSMD9 | 5715 | Proteasome (prosome, macropain) 26S subunit, non-ATPase, 9 |
58 | CDKN1C | 1028 | Cyclin-dependent kinase inhibitor 1C (p57, Kip2) |
59 | CDC2 | 983 | Cyclin-dependent kinase 1 |
60 | CCNB2 | 9133 | Cyclin B2 |
61 | CCNB1 | 891 | Cyclin B1 |
62 | CDKN1A | 1026 | Cyclin-dependent kinase inhibitor 1A (p21, Cip1) |
63 | CDK2 | 1017 | Cyclin-dependent kinase 2 |
64 | CCNA1 | 8900 | Cyclin A1 |
65 | CCNA2 | 890 | Cyclin A2 |
66 | MYC | 4609 | V-myc myelocytomatosis viral oncogene homolog (avian) |
67 | CDKN2C | 1031 | Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) |
68 | GAPDH | 2597 | Glyceraldehyde-3-phosphate dehydrogenase |
69 | ACTB | 60 | Actin, β |
70 | B2M | 567 | β-2-microglobulin |
71 | HPRT1 | 3251 | Hypoxanthine phosphoribosyltransferase 1 |
72 | OAZ1 | 4946 | Ornithine decarboxylase antizyme 1 |
73 | RPL27 | 6155 | Ribosomal protein L27 |
Downregulated expression of cell morphology genes in the Bel-7402 cells treated with (+)-terrein.
Gene name | Gene ID | Primers | Expression fold |
---|---|---|---|
FN (fibronectin) | 2335 | F, FN1: 5′-AACCTCGGCTTCCTCCATAA-3′ |
−3.61 |
VIM (vimentin) | 7431 | F, VIM: 5′-GCCAACCGGAACAATGAC-3′ |
−2.05 |
CDH2 (N-cadherin) | 1000 | F, CDH2: 5′-CTAACCCGTCGTTGCTGTTT-3′ |
−2.39 |
The Bel-7402 cells were treated with 10 μM (+)-terrein for 48 h. Each value is the mean of three independent experiments. P≤0.05 and were statistically significant. F, forward; R, reverse.
(+)-Terrein induced cell apoptosis and necrosis.
Apoptosis (%) | Necrosis (%) | |||
---|---|---|---|---|
|
| |||
Cells | Control | Treated cells | Control | Treated cells |
Bel-7402 | 5.33±1.05 | 2.26±0.50 | 1.77±0.61 | 0.83±0.23 |
A4549 | 6.17±0.93 | 0.53±0.31 | 5.43±2.55 | 1.93±1.07 |
Early apoptotic cells (%) were decreased after treatment with (+)-terrein at 10 μM to Bel-7402 or at 10 mM to A549 for 48 h. Cells not treated with (+)-terrein served as controls. Late apoptotic and necrotic cells (%) were decreased after treatment with (+)-terrein at 10 μM to Bel-7402 or at 10 mM to A549 for 48 h. Each value is the mean ± SD of five independent experiments.
Effect of (+)-terrein on the cell cycle of the Bel-7402 cells.
Sample | G0/G1 (%) | S (%) | G2/M (%) |
---|---|---|---|
Control | 59.91±2.8 | 35.29±2.1 | 4.8±0.53 |
Treated cells (10 μM) | 56.88±3.5 | 35.26±2.8 | 7.36±0.27 |
The Bel-7402 cells were treated with (+)-terrein at 10 μM for 48 h. Cells not treated with (+)-terrein served as controls. Each value is the mean ± SD of three independent experiments.
Downregulated cell-cycle genes in the Bel-7402 cells treated with (+)-terrein.
Gene no. | Gene name | Function in cell cycle | n-fold |
---|---|---|---|
1 | G1 phase and G1/S transition | −5.52 | |
2 | G1/S checkpoint | −3.30 | |
3 | Causes arrest of the cell cycle in G1 phase | −5.32 | |
4 | Cell-cycle checkpoint and cell-cycle arrest | −2.24 | |
5 | G2 phase and G2/M transition | −2.52 | |
6 | Protein kinase, membrane-associated tyrosine/threonine 1 | −2.52 | |
7 | Checkpoint kinase 2 | −2.31 | |
8 | Proliferating cell nuclear antigen | −2.10 |
Bel-7402 cells treated with 10 μM (+)-terrein for 48 h and cells not treated with (+)-terrein served as controls. Each value is the mean of three independent experiments.