2013 Volume 61 Issue 11 Pages 1156-1165
The alkaloids isolated from Stephania venosa (S. venosa) have been shown to inhibit the proliferation and to induce the apoptosis of cancer cells. However, the anti-metastatic effect of the alkaloids on cancer cell invasion is unknown. In this study, we investigated the anti-invasive properties of four alkaloids from S. venosa, crebanine (CN), O-methylbulbocapnine (OMBC), tetrahydropalmatine (THP), and N-methyltetrahydropalmatine (NMTHP), in HT1080 human fibrosacroma cells. Treatment of the cells with 15 µg/mL of CN and OMBC reduced the chemo-invasion of HT1080 cells to 45 and 50%, respectively, whereas THP and NMTHP had a negative effect. On the other hand, CN and OMBC had no effect on cell migration. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) are the extracellular matrix (ECM) degradation enzymes that play an important role in cancer cell metastasis. Results from zymography and western blot analysis showed that CN and OMBC comparatively reduced MMP-2, MMP-9, MT1-MMP and uPA expression in a dose-dependent manner. However, CN and OMBC had no effect on the activity of collagenase, MMP-2 and MMP-9. We also found that CN and OMBC reduced the nuclear translocation and DNA binding activity of nuclear factor kappa B (NF-κB), which is the expressed mediator of ECM degradation enzymes. These findings demonstrated that CN and OMBC mediated HT1080 cell invasion by the reduction of MMP-2, MMP-9, uPA and MT1-MMP expression, possibly by targeting of NF-κB signaling pathway in the HT1080 cells.
Cancer invasion and metastasis are the leading causes of morbidity and mortality in cancer patients. Tumor metastasis consists of multiple steps, including cell adhesion, invasion, migration, escape from immune system and proliferation.1) Excess extracellular matrix (ECM) degradation by proteolytic enzymes is an important step in cancer cells metastasis. The key proteases involved in degrading the components of the basement membrane are matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). The activation of these enzymes allow cancer cells access to vasculature, migration, as well as invasion into the target organ.2)
uPA is a serine protease that cleaves the ECM and converts the inactive plasminogen to active plasmin. The active plasmin can directly mediate the invasion by degrading matrix components, such as collagen, laminin and fibronectin or by activating pro-MMPs to active MMPs.3,4) Moreover, binding between uPA and its receptor uPAR also activate the intracellular signaling pathway leading to enhance cell proliferation and migration.5) MMPs are a family of zinc endopeptidases capable of breaking down the components of ECM. Among MMPs, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9 and MT1-MMP have been most strongly linked to tumor cell invasion.6,7) Therefore, inhibition of the expression and activity of ECM degradation enzymes is important in arresting cancer cell invasion and metastasis.8–10)
Since transcription factor nuclear factor kappa B (NF-κB) is a major mediator of inflammation, as well as a regulator of cancer cell development and progression. Many reports have been demonstrated that NF-κB signaling has a critical role in cancer cells metastasis.11,12) NF-κB has been shown to regulate the expression of a number of genes whose products are involved in metastasis. These include MMPs, uPA, uPAR, CXCR-4, ICAM-1 and cyclooxygenase-2 (COX-2).13) Therefore, the agent capable of suppressing NF-κB activation may be potentially useful in the inhibition of cancer cell metastasis.14,15)
Tubers of Stephania venosa (BLUME) SPRENG (S. venosa) have been used in traditional medicine in nerve tonics, aphrodisiacs, appetizers, and to treat asthma, microbial infection, hyperglycemia, malaria and cancer.16,17) This genus is also well known as a rich source of alkaloids which exhibit a variety of pharmacological activities including being an antioxidant, as well as being antiarrhythymic, and contributing to the antiproliferation of cancer.18,19) From our previous study, we demonstrated that crebanine from S. venosa induced cancer cell apoptosis and cell cycle arrest.20,21) However, the effect of the alkaloids isolated from S. venosa on the anti-invasion property has not yet been investigated.
Here, we use HT1080 human fibrosacroma cells to elucidate the potential effects of alkaloids from S. venosa on cell migration and invasion. Since the cancer cell metastasis are related to the degradation of ECM and cell motility. In this study, we investigated the influence of the active alkaloids from S. venosa on the expression of ECM degradation enzymes, uPA, MMP-2, MMP-9, MT1-MMP and the transcription factor NF-κB, in order to understand the molecular mechanism.
Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, and trypsin–ethylenediamine tetraacetic acid (EDTA) were purchased from GIBCO-BRL (Grand Island, NY, U.S.A.). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, U.S.A.). Gelatin, plasminogen and uPA were obtained from Sigma (St. Louis, MO, U.S.A.). Antibodies specific to MT1-MMP were purchased from Millipore. Antibodies specific to β-actin, poly(ADP-ribose) polymerase (PARP) and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Matrigel was purchased from Becton Dickinson (Bedford, MA, U.S.A.).
Plant MaterialThe Stephania venosa (BLUME) SPRENG (S. venosa, the Family Menispermaceae) tuber part was collected from Prachuapkhirikhan province of Thailand and identified by Forest Herbarium. A voucher specimen (BKF No. 140583) has been deposited at the Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand.
Extraction and IsolationThe alkaloids (tetrahydropalmatine (THP), crebanine (CN), O-methylbulbocapnine (OMBC), and N-methyltetrahydropalmatine (NMTHP)) were isolated and purified from the tube of S. venosa by Dr. Wilart Pompimon, as previously described21) and structure was shown in Fig. 1. Briefly, the air-dried powdered tuber of S. venosa (6.2 kg) were successively percolated with hexane and then extracted with ethyl acetate, acetone and methanol, at room temperature, respectively and followed by filtration. The alkaloid THP (3.13 g) and CN (0.17 g) were purified from the active ethyl acetate fraction (126 g), while OMBC (1.72 g) and NMTHP (0.73 g) were purified from the active acetone fraction (111.06 g) using silica gel column chromatography and the structure of the alkaloids was elucidated by extensive spectroscopic analysis (UV, IR, MS, 1H-NMR, 13C-NMR), as shown in Tables 1 and 2.21)
Cell LinesHT1080 human fibrosarcoma cells and NIH3T3 embryonic mouse fibroblast cells were maintained in DMEM, 100 U/mL penicillin and 100 µg/mL streptomycin, plus 10% FBS. The cultures were maintained in a humidified incubator with an atmosphere comprised of 95% air and 5% CO2 at 37°C. For the alkaloid treatment, alkaloids were dissolved in dimethyl sulfoxide (DMSO) and diluted with the culture medium for the final concentration of DMSO at less than 0.1%.
Cell Viability AssayHT1080 cells were plated at 2500 cells per well in 96-well plates and cultured in DMEM with 10% FBS. After being cultured for 24 h, various concentrations (0–30 µg/mL) of alkaloids were added and the samples were incubated for 24 h. At the end of the treatment, 15 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) were added and the slides were incubated for 4 h. The MTT formazan was dissolved with DMSO, and absorbance was measured using a microplate reader at 570 nm with a reference wavelength of 630 nm.
Cell Invasion and Migration AssayThe invasive and migration behaviors of HT1080 cells were tested using the modified Boyden chamber assay, as described previously.22) Briefly, polyvinylpyrrolidone-free polycarbonate filters (Millipore) with a pore size of 8 µm were coated with gelatin (0.01% w/v) for cell migration, or with Matrigel (10 µg/50 µL) for invasion assay. HT1080 cells at 1×105 cells were treated with various concentrations of alkaloids (0–15 µg/mL) and plated into the upper chamber for 18 h at 37°C in 5% CO2. The medium in the lower chamber contained a serum-free culture medium of NIH3T3 cells, which acted as a chemo-attractant. The cells that had invaded the lower surface of the membrane were fixed with methanol and stained with 1% (w/v) toluidine blue. The cells that had actively migrated to the under surface of the filter were dissolved with 20% acetic acid and indirectly quantitated by measuring the absorbance at 570 nm.
Gelatin and Casein ZymographySecretions of MMP-2 and MMP-9 of the treated cells were analyzed by gelatin zymography, as described previously.23) Briefly, HT1080 cells were treated with various concentrations (0–15 µg/mL) of alkaloids for 24 h in DMEM serum-free medium and the culture supernatant was collected in equal amounts from the cells. The culture supernatant was separated by 10% polyacrylamide gels containing 0.1% w/v of gelatin in non-reducing condition. After electrophoresis, gels were washed twice with 2.5% Triton X-100 for 30 min at room temperature to remove sodium dodecyl sulfate (SDS). The gel was then incubated at 37°C, 18 h in activating buffer (50 mM Tris–HCl, 200 mM NaCl, 10 mM CaCl2, pH 7.4). Gels were stained with Coomassie Brillant Blue R (0.1% w/v) and destained in 30% methanol with 10% acetic acid. MMPs activity appeared as a clear band against a blue background. Digestion bands were quantitated by Bio 1D software (Viber Lourmat).
The inhibitory effect of alkaloids on the activity of MMP-2 and MMP-9 was performed by gelatin zymography.24) The culture supernatant of HT1080 (MMP-2 and MMP-9) was subjected to 10% polyacrylamide gels containing 0.1% w/v of gelatin, as described above. After being washed with Triton X-100, the gel slab was cut into slices corresponding to the lanes and then put in different tanks containing various concentrations of alkaloids in activating the buffer and were then incubated at 37°C for 24 h. The strip of gel was stained with Coomassie Brillant Bule R and the MMPs activity was quantitated as described above.
The uPA secretion in culture medium was examined by casein-plasminogen zymography. The culture supernatant of the treated cells was separated by electrophoresis in 10% polyacrylamide gel electrophoresis (PAGE) copolymerized with 1 mg/mL of β-casein and 10 µg/mL human plasminogen under non-reducing conditions. After electrophoresis, gels were washed twice with 2.5% Triton X-100 for 30 min and incubated in activating buffer for 24 h. Gels were stained and destained as described above.
Collagenase Activity AssayFluorometric assay for the proteolytic activity of collagenase was performed using EnzChek Gelatinase/Collagenase Assay kit (Molecular Probe). Briefly, 1 U/mL of type IV collagenase was mixed with 10 µg/mL of fluorescein-conjugated gelatin (DQ-gelatin) containing various concentrations of alkaloids in a reaction buffer and put in 96-well microplates. The rate of proteolysis was evaluated by measuring the fluorescence intensity at 3 min interval for 30 min with a fluorometer. The fluorescence values were determined at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Enzyme activity was estimated by linear regression of the fluorescent intensity recorded during that time.
Preparation of Whole Cells Lysates, Cytoplasm and Nuclear ExtractWhole cell extracts were used to determine the expression level of MT1-MMP in HT1080 cells. Briefly, the cells were seeded at 5×105 cells per culture dish and then incubated for 24 h in DMEM with 10% FBS. The cells were treated with various concentrations of alkaloids for 24 h in DMEM serum free medium. The treated cells were washed two times with ice cold phosphate buffered saline (PBS) and extracted by incubation with lysis buffer protease inhibitor (50 mM Tris–HCl, 150 mM NaCl, 10 mM, EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL leupeptin,10 µg/mL aprotinin) for 30 min on ice. The insoluble matter was removed by centrifugation at 12000×g, 15 min and 4°C and the supernatant fraction was used to determine MT1-MMP expression.
The nuclear and cytoplasm extracts were used to determine the translocation of NF-κB from the cytoplasm to the nucleus, as previously described.22) Briefly, HT1080 cells were treated with the alkaloids for 18 h and the cells were washed twice with ice cold PBS. The cell pellets were then suspended with 400 µL of lysis buffer (10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.9, 10 mM KC1, 0.1 mM EDTA, 0.1 mM ethylene glycol bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, 1 µg/mL aprotinin). The cells were allowed to swell on ice for 20 min, after which 15 µL of 10% of Nonidet P-40 were added. The tubes were agitated on a vortex for 15 s. and centrifuged at 12000 rpm for 30 s. The supernatant was collected and represent the cytoplasm extract and the nuclear pellets were suspended in ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 2.0 pg/mL leupeptin, 2.0 µg/mL aprotinin) at an intermittent vortex for 30 min. The nuclear extract was centrifuged at 14000 rpm for 12 min and the supernatant was collected.
Western Blot AnalysisTo determine the level of protein in the whole cell lysate, cytoplasm and nuclear extract, the protein samples were separated by SDS-PAGE. After electrophoresis, the separated proteins were transferred to the nitrocellulose membrane by electro blotting. The membrane was blocked with 5% non-fat milk in PBS containing 0.5% (v/v) tween and then probed with antibodies against MT1-MMP, NF-κB (p65), PARP and β-actin. The membrane was washed and probed with the secondary antibody conjugated with horseradish peroxidase and detected by chemiluminescence (thermoscientific).
NF-κB DNA Binding Assay (Enzyme-Linked Immunosorbent Assay (ELISA))DNA binding activity of NF-κ was evaluated using NF-κB (p65) Transcription Factor Assay Kit (Cayman Chemical Company, Ann Arbor, MI, U.S.A.). Nuclear protein was extracted using NucBuster Protein Extraction Kit (Millipore). The nuclear proteins at 15 µg/mL were added to the well-coated with the dsDNA templates carrying NF-κB response element and incubated at 4°C overnight. Blank wells, positive control (provided with kit) and nonspecific binding sample were also incubated on the plate. After primary (anti-NF-κB) and secondary (goat anti-rabbit horse-radish peroxidase (HRP)) antibody treatments, developing and stopping reagent was added and absorbance was read at 450 nm. The reading for nonspecific binding was subtracted from each treatment. The percent change in activity of each test sample relative to the average of untreated samples was determined.
StatisticAll experiments were performed in triplicate. Quantifications are defined as mean±S.D. of three independent experiments and expressed as percentage of the control, which was considered to be 100%. Statistical significances of difference throughout this study were calculated by Student’s t-test (SPSS 10.0 software). A difference between the experimental groups was considered statistically significant whenever the p value is <0.01 or <0.05.
The cytotoxicity of the alkaloids from S. venosa on HT1080 cells was evaluated by MTT assay (Fig. 2). CN and OMBC showed cytotoxic effect on HT1080 cells at concentration of more than 20 µg/mL, whereas THP and NMTHP had no effect on the cancer cell growth. Therefore, an alkaloid concentration of less than 15 µg/mL (non-toxic dose) was used for cell treatments in subsequent experiments.
Cells were treated with the indicated concentrations of the alkaloids (0–30 µg/mL) for 24 h before being subjected to MTT assay. Data represent the mean±S.D. of 3 independent experiments.
To investigate the inhibitory effect of four alkaloids from S. venosa on the invasion of HT1080 cells, a Boyden chamber assay was used. The results from Fig. 3A demonstrated that CN and OMBC at 15 µg/mL reduced the invasion of HT1080 cells to 44 and 49%, respectively. However, treatment with THP and NMTHP did not effect the cell invasion. Moreover, invasion of HT1080 cells was inhibited with CN and OMBC in a dose dependent manner with an IC50 of 12 and 15 µg/mL, respectively (Fig. 3B). In contrast, CN and OMBC did not effect cell migration to the chemo-attractant through gelatin-coated filter (Fig. 3C).
HT1080 cells were seeded onto a filter coated with Matrigel containing 15 µg/mL of the alkaloids (A) or various concentrations of CN and OMBC (B) and then incubated for 18 h at 37°C. For migration assay (C), the cell was seeded onto a filter coated with gelatin containing various concentrations of CN and OMBC (0–15 µg/mL) and incubated for 18 h. Cells that actively migrated to the lower surface of the filter were quantified. Invasion and migration are expressed as a percentage of the untreated control. Data represent the mean±S.D. for 3 independent experiments. * p<0.05.
MMP-2 and MMP-9 are the key enzymes involved in ECM degradation and cancer cell invasion. To explore the suppressed invasiveness potential of CN and OMBC that was associated with MMPs secretion and activity, HT1080 cells were treated with various concentrations of CN or OMBC in the serum-free medium for 24 h and the secretion of MMP-2 and MMP-9 was analyzed by gelatin zymography. As shown in Fig. 4A, the secretion of MMP-9 was reduced by CN and OMBC in a dose dependent manner with IC50 at 11 and 15 µg/mL, respectively. Moreover, MMP-2 secretion was also significantly inhibited by CN and OMBC in a dose dependent manner. To investigate whether CN and OMBC directly inhibit the activity of MMPs and collagenase, the enzymes were directly incubated with CN and OMBC and the activity of the enzymes was detected by zymography (MMP-2 and -9) and fluorescent substrate (collagenase). As shown in Fig. 4B, CN and OMBC did not have an effect on MMP-2 and MMP-9 activity when compared with the positive control (phenanthroline at 200 µg/mL). Moreover, CN and OMBC had no effect on collagenase activity (Fig. 4C).
HT1080 cells were treated with CN and OMBC (0–15 µg/mL) for 24 h. The secretion level of MMP-2 and MMP-9 in the condition medium was determined by gelatin zymography and the band intensity was quantified by densitometey (A). For MMP-2 and MMP-9 activity assay (B), CN and OMBC (0–30 µg/mL) were directly solubilized in the activation buffer. The gel slab was cut into slices corresponding to the lanes and then put in different tanks containing different concentrations of alkaloids and 200 µg/mL of phenanthroline. The inhibitory effect of CN and OMBC on the proteolytic activity of collagenase was measured using a gelatin fluorescent substrate (C). Data represent the mean±S.D. for 3 independent experiments. * p<0.01.
To examine the influence of CN and OMBC on the secretion of uPA from HT1080 cells, the cells were treated with CN and OMBC for 24 h and uPA level in the condition medium was measured by casein zymography. As shown in Fig. 5A, the secretion of uPA was significantly reduced by CN and OMBC in a dose dependent manner with IC50 at 14 and 12 µg/mL, respectively. Moreover, the expression of MT1-MMP in HT1080 cells was reduced by CN and OMBC in a dose dependent manner (Fig. 5B).
HT1080 cells were treated with various concentrations of CN and OMBC (0–15 µg/mL) for 24 h. The culture supernatants were collected and subjected to casein zymography to determine uPA secretion levels (A). The treated cells were collected and lysated was prepared; equal amounts of proteins were loaded and actin was used as the protein loading control. The level of MT1-MMP was determined by Western blot analysis (B). The intensity of protein band was quantified by densitometry. The data are representative of 3 independent experiments with * p<0.01.
There is a lot of evidence that NF-κB play a key role in the expression of MMPs and uPA. Therefore, we analyzed the NF-κB DNA binding activity of HT1080 cells to determine the effect of CN and OMBC at the transcription level of MMPs and uPA. As shown in Fig. 6A, treatment the cells with CN and OMBC at 10 µg/mL significant reduced NF-κB DNA binding activity. Furthermore, the expression of p65 (NF-κB) in nucleus and cytoplasm extract was determined by Western blot to assess the possible inhibitory effect of CN and OMBC on NF-κB translocation from cytoplasm to nucleus. Western blot analysis revealed that the treatment of CN and OMBC reduced the p65 level in the nucleus in a dose dependent manner when compared with the untreated cells, as show in Fig. 6B.
HT1080 cells were treated with the indicated concentrations of CN and OMBC for 24 h. Nuclear and cytoplasmic extracts were prepared. (A) DNA binding activity of NF-κB was determined from nuclear extract by ELISA for each sample relative to vehicle control (100%). (B) Nuclear and cytoplasm extract was subjected to SDS-PAGE, followed by Western blot analysis with anti-p65(NF-κB) antibody. PARP and β-actin were used as the nuclear and cytoplasmic protein loading controls, respectively. Data from a typical experiment are depicted and similar results were obtained in 3 independent experiments with * p<0.01.
Recently, we isolated alkaloids from S. venosa including NMTHP, THP, CN and OMBC, and the anti proliferation activity they displayed against a variety of cancer cells was reported. Among those compounds, CN presented the most proliferation inhibition.21) Moreover, we also reported that CN induced leukemic cell apoptosis via intrinsic and extrinsic pathways.20) On the other hand, THP and CN has have been reported to have multidrug resistant reversing properties in human breast cancer cells.25) To date, there is no evidence to show that alkaloids from S. venosa exert an effect on cancer cell metastasis. Here, we investigated the anti-metastasis properties of two alkaloid groups, protoberberine (THP and NMTHP) and aporphine (CN and OMBC), from S. venosa in HT1080 cells.
Metastasis has been found to be accompanied by various physiological alterations involved in the degradation of ECM, which allowed cancer cells to invade the blood or the lymphatic vessel and travel to other tissues or organs.26) The present study is the first report to demonstrate that non-cytotoxic doses of aporphine (CN and OMBC), but not protoberberine (THP and NMTHP), significantly reduced HT1080 cell invasion and passed though the basement membrane. However, CN and OMBC had no effect on cell migration.
Numerous studies have indicated that the inhibition of MMPs expression or the enzyme activity can be used as early targets in preventing cancer metastasis. MMP-2 and MMP-9 are recognized to be therapeutic targets of anti-cancer drugs due to their degrading action of both enzymes on collagen type IV, which are major components of the basement membrane.27) Our result from gelatin zymography showed that CN reduced MMP-2 and MMP-9 secretion more than OMBC. However, CN and OMBC had no effect on MMP-2, MMP-9, and collagenase activity when gelatin zymography and DQ-gelatin were used. Moreover, CN and OMBC also reduced the expression of the pro-MMP-2 activator, MT1-MMP, in a dose dependent manner, which suggested that CN and OMBC reduced HT1080 cell invasion via a reduced expression and secretion of MMPs.
The uPA plays an important role in the degradation of basement membranes, while the activation of the uPA/uPAR/plasmin proteolytic network plays a key role in tumor invasion and the dissemination of various malignancies.28) It is well documented that high concentrations of uPA and uPAR in various cancers types, including breast and ovarian cancer, is a strong indicator of poor prognostic.29,30) This study showed that CN and OMBC treatment of HT1080 cells comparably reduced uPA secretion in a dose dependent manner. These data collectively support the inhibitory effect of CN and OMBC on cancer invasion via reduced ECM degradation.
NF-κB is a transcription factor that promotes tumorigenesis and cancer cell invasion and metastasis.31) Reduction of NF-κB activation is effective in the prevention and treatment of cancer.32) As is commonly known, the active NF-κB consists of a REL family/p65 subunit and a p50 or p52 subunit. NF-κB is maintained in the cytoplasm in an inactive form, bound by an inhibitory protein called an inhibitor of κB (IκB). NF-κB activation involves its release from IκBα and the subsequent translocation from cytoplasm to the nucleus, where it binds to cognate sequences in the promoter region of many targeted genes, including VEGF, uPA, MMP-9, MT1-MMP, ICAM-1 and COX-2.33–35) Therefore, NF-κB DNA binding activity and translocation from cytoplasm to nucleus are hallmark of its activation. In the present study, we demonstrated that CN and OMBC reduced NF-κB DNA binding activity. Moreover, the translocation of NF-κB from the cytoplasm to the nucleus in HT1080 cells was inhibited by CN and OMBC. Hence, the inhibitory effect of CN and OMBC on NF-κB may be involved in the anti-invasion mechanism of CN and OMBC.
Among four alkaloids from S. venosa, CN presented the most anti-invasive effect, whereas the dislocation of dimethoxy on ring D from C-8 and C-9 to C-10 and C-11 of OMBC showed a faintly lesser effect than its isomer. These results are comparable with MMP-2, MMP-9, MT1-MMP, and the uPA expression level. However, an anti-invasive effect was not observed in protoberberine alkaloids (THP and OMTHP) treated cells. These results contrast with previous studies which demonstrated that berberine exhibited anti-metastasis and anti-cancer in various cancer cell lines.36,37) THP and berberine are protoberberine alkaloids and differ only by the presence or absence of the 1,3-benzodioxole group. Thus, we suggest that a loss of the 1,3-benzodioxole group from berberine in THP and OMTHP, may cause a loss of anti-metastasis activity.
In conclusion, our data for the first time indicated that CN and OMBC could inhibit invasion of HT10080 cells. Characterization of the detailed mechanism of the inhibitory effect of CN and OMBC revealed a reduction of ECM degradation enzymes due, in part, to reduced NF-κB activation. CN and OMBC should be considered as a possible therapeutic agent for inhibiting the metastasis and invasion of cancer cells. Further investigations will be required to assess the potential of CN and OMBC in the treatment of cancer.
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| Tetrahydropalmatine (1) | Crebanine (2) | O-Methylbulbocapnine (3) | N-Methyltetrahydropalmatine (4) | ||||||||
| Position | δ1H (J Hz) | δ13C (DEPT) | Position | δ1H (J Hz) | δ13C (DEPT) | Position | δ1H (J Hz) | δ13C (DEPT) | Position | δ1H (J Hz) | δ13C (DEPT) |
| 1 | 6.73 (s) | 108.59 (CH) | 1 | — | 142.00 (C) | 1 | — | 144.66 (C) | 1 | 6.79 (s) | 109.73 (CH) |
| 2 | — | 147.48 (C) | 2 | — | 146.50 (C) | 2 | — | 150.06 (C) | 2 | — | 149.62 (C) |
| 3 | — | 147.42 (C) | 3 | 6.52 (s) | 106.78 (CH) | 3 | 6.70 (s) | 107.81 (CH) | 3 | — | 148.64 (C) |
| 4 | 6.62 (s) | 111.33 (CH) | 3a | — | 126.50 (C) | 3a | — | 120.73 (C) | 4 | 6.69 (s) | 111.50 (CH) |
| 4a | — | 126.76 (C) | 4 | (a) 3.13 (obsc) | 29.14 (CH2) | 4 | (a) 3.30 (obsc) | 26.93 (CH2) | 4a | — | 119.22 (C) |
| 5 | (a) 3.13 (obsc) | 29.06 (CH2) | (b) 2.62 (m) | (b) 2.96 (dd, 17.39, 3.53) | 5 | (a) 3.30 (obsc) | 23.27 (CH2) | ||||
| (b) 2.66 (obsc) | 5 | (a) 3.05 (obsc) | 53.59 (CH2) | (b) 3.16 (dd, 18.15, 6.86) | |||||||
| 6 | (a) 3.21 (obsc) | 51.48 (CH2) | (b) 2.51 (t,d, | 5 | (a) 3.80 (obsc) | 53.94 (CH2) | |||||
| (b) 2.66 (obsc) | 11.63, 3.69) | (b) 3.54 (obsc) | 6 | (a) 3.82 (obsc) | 51.86 (CH2) | ||||||
| 8 | (a) 4.24 (d, 15.63) | 53.96 (CH2) | 6a | 3.05 (obsc) | 61.85 (CH) | 6a | 4.34 (obsc) | 69.11 (CH) | (b) 3.67 (obsc) | ||
| (b) 3.54 (d,15.63) | 6b | — | 126.55 (C) | 6b | — | 125.04 (C) | 8 | 4.94 (s) | 59.83 (CH2) | ||
| 8a | — | 127.70 (C) | 7 | (a) 3.67 (dd, | 26.89 (CH2) | 7 | (a) 3.83 (obsc) | 25.41 (CH2) | 8a | — | 119.92 (C) |
| 9 | — | 145.06 (C) | 14.66, 4.3) | (b) 2.60 (m) | 9 | — | 145.58 (C) | ||||
| 10 | — | 150.24 (C) | (b) 2.29 (m) | 7a | — | 126.77 (C) | 10 | — | 151.24 (C) | ||
| 11 | 6.79 (d, 8.38) | 110.94 (CH) | 7a | — | 129.76 (C) | 8 | 7.87 (d, 8.72) | 124.81 (CH) | 11 | 6.87 (d, 8.35) | 113.35 (CH) |
| 12 | 6.88 (d, 8.38) | 123.82 (CH) | 8 | — | 151.98 (C) | 9 | 7.03 (d, 8.72) | 112.61 (CH) | 12 | 6.82 (d, 8.35) | 123.32 (CH) |
| 12a | — | 128.61 (C) | 9 | — | 145.80 (C) | 10 | — | 153.94 (C) | 12a | — | 121.16 (C) |
| 13 | (a) 3.27 (dd, 15.76, 3.65) | 36.28 (CH2) | 10 | 6.87 (d, 8.47) | 110.20 (CH) | 11 | — | 147.03 (C) | 13 | (a) 3.45 (d, 2.59) | 34.00 (CH2) |
| 11 | 7.80 (d, 8.47) | 123.05 (CH) | 11a | — | 124.44 (C) | (b) 3.00 (dd, | |||||
| (b) 2.83 (dd, 15.76, 11.49) | 11a | — | 124.59 (C) | 11b | — | 117.60 (C) | 18.35, 10.12) | ||||
| 11b | — | 116.46 (C) | 12 | (a) 6.15 (d, 0.74) | 102.86 (CH2) | 13a | 5.09 (obsc) | 64.98 (CH) | |||
| 13a | 3.54 (obsc) | 59.29 (CH) | 12 | (a) 6.06 (d,1.44) | 100.56 (CH2) | (b) 6.00 (d, 0.74) | 13b | — | 123.93 (C) | ||
| 13b | — | 129.66 (C) | (b) 5.91 (d,1.44) | N‒CH3 | 3.21 (s) | 42.12 (CH3) | N–CH3 | 3.40 (s) | 49.98 (CH3) | ||
| 2-OCH3 | 3.87 (s) | 56.05 (CH3) | N–CH3 | 2.60 (s) | 43.95 (CH3) | 10-OCH3 | 3.90 (s) | 56.30 (CH3) | 2-OCH3 | 3.85 (s) | 56.03 (CH3) |
| 3-OCH3 | 3.89 (s) | 55.84 (CH3) | 8-OCH3 | 3.81 (s) | 60.65 (CH3) | 11-OCH3 | 3.86 (s) | 61.19 (CH3) | 3-OCH3 | 3.86 (s) | 56.18 (CH3) |
| 9-OCH3 | 3.85 (s) | 60.14 (CH3) | 9-OCH3 | 3.90 (s) | 55.71 (CH3) | 9-OCH3 | 3.84 (s) | 60.84 (CH3) | |||
| 10-OCH3 | 3.84 (s) | 55.81 (CH3) | 10-OCH3 | 3.82 (s) | 55.88 (CH3) | ||||||
 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Tetrahydropalmatine (1) | Crebanine (2) | O-Methylbulbocapnine (3) | N-Methyltetrahydropalmatine (4) | ||||||||
| Position | HMBC correlation | COSY correlation | Position | HMBC correlation | COSY correlation | Position | HMBC correlation | COSY correlation | Position | HMBC correlation | COSY correlation |
| 1 | C-2, C-4a, C-13b | — | 1 | — | — | 1 | — | — | 1 | C-2, C-3, C-4a, C-13b | — |
| 2 | — | — | 2 | — | — | 2 | — | — | 2 | — | — |
| 3 | — | — | 3 | C-1, C-2, C-3a, | — | 3 | C-1, C-2, C-3a, C-4 | — | 3 | — | — |
| 4 | C-1, C-3, C-5, C-13b | — | C-4,C-11b | 3a | — | — | 4 | C-2, C-3, C-4a, C-5 | — | ||
| 4a | — | — | 3a | — | — | 4 | (a) C-5, C-6b | H-4b, H-5a | 4a | — | — |
| 5 | (a) C-4a | H-6b, H-5b | 4 | (a) C-3a, C-5, C-6b | H-4b | (b) C-3, C-3a, C-6b | H-4a, H-5b | 5 | (a) C-4a | H-5b, H-6a | |
| (b) C-4a | H-5a, H-6a | (b) C-3, C-3a, C-6b | H-4a | 5 | (a) C-6b | H-4a, H-5b | (b) C-4a | H-5a, H-6b | |||
| 6 | (a) C-4a, C-5, C-8 | H-6b | 5 | (a) C-3a, C-4, C-6a, | H-5b | (b) — | H-4b, H-5a | 6 | (a) — | H-5a, H-6b | |
| (b) C-4a, C-13a | H-6a, H-5a | N–CH3 | 6a | — | H-7b | (b) — | H-5b, H-6a | ||||
| 8 | (a) C-6, C-8a, C-9, | H-8b | (b) C-3a, C-4, C-6a, | — | 6b | — | — | 8 | C-6, C-8a, C-9, C-12a, | — | |
| C-12a, C-13a | N–CH3 | 7 | (a) — | H-7b | C-13a, C-13b, N–CH3 | ||||||
| (b) C-6, C-13a | H-8a | 6a | C-6b, N–CH3 | — | (b) C-7a | H-6a, H-7a | 8a | — | — | ||
| 8a | — | — | 6b | — | — | 7a | — | — | 9 | — | — |
| 9 | — | — | 7 | (a) C-6a, C-7a, C-9, | H-7b | 8 | C-7, C-7a, C-9, | H-9 | 10 | — | — |
| 10 | — | — | C-11a | C-10, C-11, C-11b | 11 | C-9, C-10, C-12a, | H-12 | ||||
| 11 | C-8a, C-9, C-10, C-12 | H-12 | (b) C-6a, C-7a, C-8, | H-7a | 9 | C-10, C-11, C-11a | H-8 | 12 | C-8a, C-9,C-10, C-13, | H-11 | |
| 12 | C-9, C-10, C-12a, C-13 | H-11 | C-9,C-10, C-11, C-11a | 10 | — | — | C-13a | ||||
| 12a | — | — | 7a | — | — | 11 | — | — | 12a | — | — |
| 13 | (a) C-8a, C-12, C-13a | H-13b | 8 | — | — | 11a | — | — | 13 | (a) C-12a | H-13b, H-13a |
| (b) C-8a, C-12a, C-13a | H-13a, H-13a | 9 | — | — | 11b | — | — | (b) C-12a, C-13a | H-13a, H-13a | ||
| 13a | C-6, C-12a | H-13b | 10 | C-7a, C-8, C-9, C-11a | H-11 | 12 | (a) C-1, C-2 | — | 13a | C-13b | H-13b, H-13a |
| 13b | — | — | 11 | C-7a, C-8, C-9, C-10, C-11b | H-10 | (b) C-1, C-2 | — | 13b | — | — | |
| 2-OCH3 | C-2, C-3 | — | — | N–CH3 | — | — | N–CH3 | C-6, C-8 | — | ||
| 3-OCH3 | C-2, C-3 | — | 11a | — | — | 10-OCH3 | C-9, C-10 | — | 2-OCH3 | C-2 | — |
| 9-OCH3 | C-9 | — | 11b | (a) C-1, C-2 | — | 11-OCH3 | C-11 | — | 3-OCH3 | C-3 | — |
| 10-OCH3 | C-10 | — | 12 | (b) C-1, C-2 | — | 9-OCH3 | C-9 | — | |||
| C-4, C-5 | — | 10-OCH3 | C-10 | — | |||||||
| N–CH3 | C-8 | — | |||||||||
| 8-OCH3 | C-9, C-10 | — | |||||||||
| 9-OCH3 | — | ||||||||||
This work was supported by the Thailand Research Fund (MRG. No. 5480293), and the Faculty of Medicine Research Fund, Faculty of Medicine, Chiang Mai University.
