To examine the effect of Xuefuzhuyutang on the metastasis of cancer, the following experiments were carried out. Before the main experiments, the cytotoxicity was measured by putting Xuefuzhuyutant sample in HT1080. Then zymography was made to examine the change of gelatinolytic activity. Western blotting was carried out to examine the changes of Fos, Jun, Ets, Erk, md JNK. In vitro invasion assay with transwells coated by collagen and matrigel was carried out. From the above results the following conclusions were obtained. 1. The experimental result about cytotoxicity of Xuefuzhuyutang agaitst HT1080 was a below. The stained cell count after being treated by by Xuefuzhuyutang sample $400{\mu}g/ml$ for 24 hours was 0.9% of total cells, and the stained cell count by Xuefuzhuyutang sample $100{\mu}g/ml$ was 1.5% of total cells. Both were near the level of control group which showed 0.6% stained. 2. The result of collagenase assay was as below. In Xuefuzhuyutang sample $400{\mu}g/ml$, MMP2 was reduced as compared with TPA control group, and the band of MMP-9 induced by TPA disapappeared. In Xuefuzhuyutang samle $800{\mu}g/ml$ both bands of MMP-2 and MMP-9 disapeared. 3. The results of western blots for Jun, Fos, Ets, Erk, JNK were a below. In Xuefuzhuyutang sample $200{\mu}g/ml$, Ets was reduced, and Jun, Fos were increased. 4 The result of invasion assay was as below. The number of cells which migrated across transwell membrane in Xuefuzhuyutang-treated group was less than that of control(+TPA) group. From the above results, it was concluded that Xuefuzhuyutang might inhibit the activity of collagenase not by the MMP-2, MMP-9 promoter but by the other way.
To examine the effect of Shiquandabutang on the metastasis of cancer, the following experiments were made. Before the main experiments, the cytotoxicity was measured by putting Shiquandabutang sample in HT1080. Then zymography was made to examine the change of gelatinolytic activity. And western blotting was carried out to examine the changes of Fos, Jun, Ets, the transcription factors of MMP-2, MMP-9, and Erk, JNK on signal transduction pathway to AP-1. Third, in vitro invasion assay with transwells coated by collagen and matrigel was carried out. From the results of the above the following conclusions were obtained. 1. The experimental result about cytotoxicity of Shiquandabutang against HT1080 was as below. The stained cell count after being treated by Shiquandabutang sample $400{\mu}g/ml$ for 24 hours was 0.9% of total cells, and the stained cell count by Shiquandabutang sample $100{\mu}g/ml$ was 1.5% of total cells. Both were near the level of control group which showed 0.6% stained. 2. The result of collagenase assay was as below. In Shiquandabutang sample $400{\mu}g/ml$, MMP-2 was reduced as compared with TPA control group, and the band of MMP-9 induced by TPA disappeared. In Shiquandabutang sample $800{\mu}g/ml$, both bands of MMP-2 and MMP-9 disappeared. 3. The results of western blots for Jun, Fos, Ets, Erk, JNK were as below. In Shiquandabutang sample $200{\mu}g/ml$, Ets was reduced, and Fos were increased. 4. The result of invasion assay was as below. The number of cells which migrated across transwell membrane in Shiquandabutang-treated group was less than that of +TPA control group. From the above results, it was concluded that Shiquandabutang might control the appearing and acting of collagenase not by the MMP-2, -9 promoter but by other way.
Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive tumor cells to mimic the pattern of embryonic vasculogenesis, which was associated with invasion and metastasis. The grape seed proanthocyanidins (GSPs) had attracted much attention as a potential bioactive anti-carcinogenic agent. However, GSPs regulation of VM and its possible mechanisms in a triple-negative breast cancer cells (TNBCs) remain not clear. Therefore, we examined the effect of GSPs on VM information in HCC1937 cell model. In this study, we identified the VM structure via the three-dimensional (3D) matrix in vitro. Cell viability was measured using the CCK8 assay. The effects of GSPs on human triple-negative breast cancer cells (TNBCs) HCC1937 in terms of related proteins of VM information were determined using western blot analysis. In vitro, the tubular networks were found in highly invasive HCC1937 cells but not in the non-invasive MCF-7 cells when plated on matrigel. The number of vascular channels was significantly reduced when cells were exposed in GSPs ($100{\mu}g$/ml) and GSPs ($200{\mu}g/mL$) groups (all p<0.001). Furthermore, we found that treatment with GSPs promoted transition of the mesenchymal state to the epithelial state in HCC1937 cells as well as reducing the expression of Twist1 protein, a master EMT regulator.GSPs has the ability to inhibit VM information by the suppression of Twist1 protein that could be related to the reversal of epithelial-to-mesenchymal (EMT) process. It is firstly concluded that GSPs may be an p otential anti-VM botanical agent for human TNBCs.
Objective: The aim of this study was to investigate the clinical significance of annexin a1 (ANXA1) and provide molecular evidence to support that decreased ANXA1 expression could enhance cancer migration and invasion in pancreatic ductal adenocarcinoma (PDAC). Materials and Methods: Immunohistochemistry of a tissue microarray with 162 surgically resected PDAC specimens was performed to examine the expression of ANXA1. We also investigated the relationship between ANXA1 expression and clinicopathological factors and prognosis of PDAC patients. We further studied the role of ANXA1 in PDAC cell proliferation, migration and invasion by cell proliferation assay, migration assay and matrigel invasion assay with reduced ANXA1 expression by RNAi. Western blotting was used to detect matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression. We also detected MMP-9 enzyme activity by gelatin zymography. Results: Decreased expression of ANXA1 was significantly associated with poor differentiation, lymph node metastasis and advanced TNM stage of PDAC patients (p<0.05). Moreover, decreased expression of ANXA1 was correlated with poor survival (p<0.05). Furthermore, we found that ANXA1 knockdown inhibited cell proliferation, induced G1 phase cell cycle arrest, increased PDAC cell migration and invasion capacity compared with controls. In addition, Western blotting showed that ANXA1 knockdown increased the MMP-9 protein level and decreased TIMP-1 expression. Gelatin zymography showed that MMP-9 enzyme activity was also elevated. Conclusions: Negative ANXA1 expression is a most unfavorable prognostic factor for PDAC patients. ANXA1 knockdown inhibits cell proliferation by inducing G1 phase cell cycle arrest and increases migration and invasion of PDAC cells through up-regulating MMP-9 expression and activity, implying that ANXA1 may serve as a promising prognostic biomarker and therapeutic target for PDAC.
Aims and Background: Prostate cancer is one of the most common malignant tumors in the male reproductive system, which causes the second most cancer deaths of males, and control of angiogenesis in prostate lesions is of obvious importance. This study assessed the effect of apogossypolone (ApoG2) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs). Subjects and Methods: HUVECs were treated with different concentrations of ApoG2. The survival rate of HUVECs were determined by MTT assay. Utrastructural changes of HUVECs were assessed with transmission electron microscopy. Apoptosis in HUVECs was analyzed by flow cytometry and cell migration by Boyden chamber assay. Matrigel assays were used to quantify the development of tube-like networks. Results: ApoG2 significantly inhibited HUVEC growth even at 24 h (P<0.05). The inhibitory effect of ApoG2 is more obvious as the concentration and the culture time increased (P<0.05). These results indicate that ApoG2 inhibits the proliferation of HUVECs in a time- and concentration-dependent manner with increase of the apoptosis rate. Besides, ApoG2 reduced the formation of total pseudotubule length and network branches of HUVECs. Conclusions: The results suggest that ApoG2 inhibits angiogenesis of HUVECs by growth inhibition and apoptosis induction.
Park, Un-Chul;Cho, Myung-Soo;Park, Jung-Hyun;Kim, Sang-Jin;Ku, Seung-Yup;Choi, Young-Min;Moon, Shin-Yong;Yu, Hyeong-Gon
Clinical and Experimental Reproductive Medicine
/
v.38
no.4
/
pp.216-221
/
2011
Objective: To differentiate the human embryonic stem cells (hESCs) into the retinal pigment epithelium (RPE) in the defined culture condition and determine its therapeutic potential for the treatment of retinal degenerative diseases. Methods: The embryoid bodies were formed from hESCs and attached on the matrigel coated culture dishes. The neural structures consisting neural precursors were selected and expanded to form rosette structures. The mechanically isolated neural rosettes were differentiated into pigmented cells in the media comprised of N2 and B27. Expression profiles of markers related to RPE development were analyzed by reverse transcription-polymerase chain reaction and immunostaining. Dissociated putative RPE cells ($10^5$ cells/5 ${\mu}L$) were transplanted into the subretinal space of rat retinal degeneration model induced by intravenous sodium iodate injection. Animals were sacrificed at 1, 2, and 4 weeks after transplantation, and immnohistochemistry study was performed to verify the survival of the transplanted cells. Results: The putative RPE cells derived from hESC showed characteristics of the human RPE cells morphologically and expressed molecular markers and associated with RPE fate. Grafted RPE cells were found to survive in the subretinal space up to 4 weeks after transplantation, and the expression of RPE markers was confirmed with immunohistochemistry. Conclusion: Transplanted RPE cells derived from hESC in the defined culture condition successfully survived and migrated within subretinal space of rat retinal degeneration model. These results support the feasibility of the hESC derived RPE cells for cell-based therapies for retinal degenerative disease.
Pluripotent stem cells (PSCs) have lots of potential in biomedical sciences owing to its potential to differentiate into any kind of cells in the body. However, it is still a challenge to culture PSCs on a large scale for application to regenerative medicine. Herein, we introduce a synthetic polymer that enables large-scale suspension culture of human PSCs. By employing suspension culture, it became unnecessary to use conventional substrata such as mouse embryonic fibroblast (MEF) or Matrigel$^{TM}$, which are believed to be main causative sources of xenogeneic contamination in cultured human PSCs in vitro. Human PSCs were cultured in the medium in which elastin-like polypeptide (ELP) dissolved. The ELP in the medium became harden as temperature increases by transforming the medium into a semi-solid gel that supported growth of human PSCs in suspension. Gel-sol transition temperature of ELP can be adjusted by modifying the peptide sequence in which 5 amino acids, Val-Pro-Gly-Xaa-Gly, repeated sequentially. We constructed 3D suspension media having transition temperature around $33{\sim}35^{\circ}C$ using an ELP consisted of 40, 60, or 80 repeats of a monomer, which was Val-Pro-Gly-Val-Gly. Among the ELPs, ELP80 was chosen as the best ELP to support growth of human PSCs in suspension culture. This result suggests that the ELP80 can be a medium component for culturing human PSCs in large-scale.
A new piperazine derivative, 8J-8029, is a synthetic anti-cancer agent which exhibits both microtubule and topoisomerase II inhibiting activities. In this study, we investigated the ability of 8J-8029 for anti-angiogenesis and apoptosis. 8J-8029 decreased the bFGF-induced angiogenesis in the CAM and the mouse Matrigel implants, in vivo. 8J-8029 inhibited the proliferation, migration, invasion, tube fonnation, and expression of MMP-2 in BAECs. In addition, 8J-8029 reduced the cell viability in HepG2 cells, caused the production of fragmented DNA and the morphological changes corresponding to apoptosis. 8J-8029 also elicited the release of cytochrome c and the activation of caspase-3. Taken together, these results suggest 8J-8029 may be a candidate for anti-cancer agent with the ability to inhibit the angiogenesis of endothelial cells and to induce the apoptosis of tumor cells.
Background: Platycodin D (PD), a triterpenoid saponin isolated from the Chinese medicinal herb Platycodonis radix, possesses anti-cancer effects in several cancer cell lines. The aim of this study was to evaluate its anticancer activities in hepatocellular carcinoma cells. Materials and Methods: MTT and colony formation assays were performed to evaluate cell proliferation, along with flow cytometry and Western blotting for apoptosis. Cell adhesion was tested by observing cellular morphology under a microscope, while the transwell assay was employed to investigate the cell migration and invasion. Results: PD concentration-dependently inhibited cell proliferation in both HepG2 and Hep3B cells, and significantly suppressed colony formation and induced apoptosis in HepG2 cells. The protein levels of cleaved poly ADP-ribose polymerase (PARP) and Bax were up-regulated while that of survivin was down-regulated after treatment with PD. Moreover, PD not only obviously suppressed the adhesion of HepG2 cells to Matrigel, but also remarkably depressed their migration and invasion induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). Conclusions: PD presents anti-cancer potential in hepatocellular carcinoma cells via inducing apoptosis, and inhibiting cell adhesion, migration and invasion, indicating promising features as a lead compound for anti-cancer agent development.
A culture system for lactating rat mammary acini was evaluated, where the primary indicator of performance was lactose secretion, measured by a sensitive bioluminescence assay. Lactose secretion was reduced by half (p<0.01) over the first 6 h of culture by overnight feed withdrawal (FW) from tissue donors but was sensitive to increased glucose concentration in the culture media (p<0.001) up to 30 mM. Lactose production of cells from fed donors over the first 6 h in culture in 30 mM glucose was 8.9 fmol/cell/h - a rate calculated to be about half that in vivo. No significant difference was shown in lactose secretion by cells from fed or FW rats over 6-24 h. Lactose secretion was 3.6 fmol/cell/h by cells from fed animals in 40 mM glucose concentration media over the 6-24 h culture period. Addition of insulin to the culture media had no effect on rates of lactose secretion while addition of prolactin and hydrocortisone, with or without insulin, significantly (p<0.001) decreased lactose production over both 0-6 h and 6-24 h culture periods. Lactose synthesis in vitro was significantly enhanced by aeration of the media during collagenase digestion of mammary tissue (p<0.05). No improvement in lactose secretion was effected by shaking of cells during culture, Matrigel coating of culture dishes or change in cell density over a range up to 2.5 million cells per ml.
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