Vincosamide Has a Function for Inhibiting Malignant Behaviors of Hepatocellular Carcinoma Cells.

Background: Vincosamide (Vinco) was first identified in the methanolic extract of the leaves of Psychotria leiocarpa, and Vinco has important anti-inflammatory effects and activity against cholinesterase, Vinco also has a trait to anti-tumor. However, whether Vinco can inhibit the malignant behaviors of hepatocellular carcinoma (HCC) cells is still unclear. In the present study, we explored the role of Vinco in suppressing the malignant behaviors of HCC cells. Methods: MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide), trypan blue exclusion assay, the Cell Counting Kit (CCK)-8 and flow cytometric analysis were applied to detect the proliferation and apoptosis of HCC cells; electron microscopy was performed to observe the change of cellular mitochondrial morphology; scratch repair and Transwell assays were used to analyze the migration and invasion of HCC cells; expression and localization of proteins were detected by laser confocal microscopy and Western blotting; the growth of the cancer cells in vivo was assessed in a mouse tumorous model. Results: At a dose of 10 - 80 µg/mL, Vinco inhibited the proliferation, migration, invasion and promoted apoptosis of HCC cells in a dose-dependent manner but had low cytotoxicity effect on normal liver cells. Additionally, 80 µg/mL of Vinco could significantly disrupt the morphology of mitochondria, suppress the migration and invasion of HCC cells. The growth of HCC cells in the animal tumorous model was significantly inhibited after treatment with Vinco (10 mg/kg/day) for 3 days. The results of the present study indicated that Vinco (10 - 80 µg/mL) played a role in activating caspase-3, promoting the expression of phosphate and tension homology deleted on chromosome 10 (PTEN), and inhibiting the phosphorylation of AKT (Ser473) and mTOR (Thr2448); Vinco also has a trait for suppressing the expression of CXCR4, Src, MMP9, EpCAM, Ras, Oct4 and cancer stem cell "stemness markers" CD133 and CD44 in HCC cells. Conclusions: Vinco has a role in inhibiting the malignant behaviors of HCC cells; the role molecular mechanism of Vinco may be involved in restraining expression of the growth-, metastasis-related factors, such as Src, Ras, MMP9, EpCAM, CXCR4; activating the activity of caspase-3 and blocking PI3K/AKT signaling pathway. Thus, Vinco should be considered as a new chemotherapy agent for HCC patients.

Nondiagnostic cytological results on ultrasound-guided fine needle aspiration: Does the thyroid nodule depth matter?

OBJECTIVE: To evaluate whether thyroid nodule depth and other ultrasound features can predict nondiagnostic cytological results on ultrasound-guided fine needle aspiration. MATERIAL AND METHODS: This retrospective study included 412 thyroid nodules that underwent ultrasound-guided fine needle aspiration from 2014 to 2015. The nodules were classified as nondiagnostic and diagnostic by the cytopathological results. Clinical information of the patients (ie., age, sex) and ultrasound features (ie., size, depth, calcification, cystic content, vascularity) of the nodules were recorded and compared between the nondiagnostic group and diagnostic group. RESULTS: Age and sex were not significantly different between the nondiagnostic group and diagnostic group (P > 0.05). Nodule depth >15 mm (OR, 7.128; P < 0.001), peripheral rim calcification (OR, 5.725; P = 0.01) and cystic content >50% (OR, 2.995; P = 0.018) were factors for the nondiagnostic ultrasound-guided fine needle aspiration cytopathological results. Macrocalcification in the nodule sized 5-10 mm was associated with the nondiagnostic results (P = 0.04). Nodule size and vascularity were not associated with the nondiagnostic results (P > 0.05). CONCLUSIONS: Nodule depth >15 mm, peripheral rim calcification and cystic content >50% were three independent predictors of the nondiagnostic cytopathological results. Macrocalcification in the nodule sized 5-10 mm was also associated with the nondiagnostic results.

[Effects of alpha-fetoprotein on the expression of TRAIL death receptor-2 and its role on resisting the cytotoxicity of TRAIL in hepatoma cells].

OBJECTIVE: To explore the mechanism of Alpha-fetoprotein (AFP) effects on hepatocellular carcinoma cells (HCC) resistances apoptosis induced by tumor necrosis factor-related apoptosis inducing-ligand (TRAIL). METHODS: The expressed alteration of TRAIL receptor-2 (DR5) after the human hepatoma cells line Bel 7402 (AFP-producing) and HLE cells (non-AFP producing) were treated with all trans retinoic acid (ATRA) were determined by Western blot; Interaction of AFP with RAR-beta was analyzed by co-immunoprecipitation (Co-IP); Laser confocal microscopy was used to observe co-localization of AFP and RAR-beta; Short small RNA interfering (RNAi) was applied to knock down the expression of AFP in Bel 7402 cells; The full AFP gene cDNA was inserted into pcDNA3.1 vector and constructed the expressed vector of AFP (named pcDNA3.1-afp); The growth of hepatoma cells was analyzed by MTT. RESULTS: Bel 7402 and HLE cells expressed DR5, lowed dosage of ATRA (40mumol/L) had no influence on the expression of DR5 in Bel 7402 cells, but ATRA (160mumol/L) could inhibit the expression of AFP and promote the expression of DR5 significantly; Co-IP indicated that AFP had a property for interacting with RAR-beta; The results also demonstrated AFP co-localization with RAR-beta in cytoplasm of Bel 7202 cells; The expression of DR5 was enhanced while the expression of AFP was knocked down by RNAi. pcDNA3.1-afp vector was transfected into HLE cells, the growth of HLE cells were stimulated and TRAIL cytotoxicity of HLE cells were reduced. But when the expression of AFP was knocked down the sensitivity of Bel 7402 cells to TRAIL was enhanced. CONCLUSIONS: These data provided that AFP had a capability to interact with RAR-beta and suppressed the expression of DR5. AFP could play pivotal role on hepatoma cells resistance-induced apoptosis by TRAIL.

Alpha-fetoprotein triggers hepatoma cells escaping from immune surveillance through altering the expression of Fas/FasL and tumor necrosis factor related apoptosis-inducing ligand and its receptor of lymphocytes and liver cancer cells.

AIM: To investigate the mechanism of alpha-fetoprotein (AFP) in escaping from the host immune surveillance of hepatocellular carcinoma. METHODS: AFP purified from human umbilical blood was administrated into the cultured human lymphoma Jurkat T cell line or hepatoma cell line, Bel7402 in vitro. The expression of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and its receptor (TRAILR) mRNA were analyzed by Northern blot and Western blot was used to detect the expression of Fas and Fas ligand (FasL) protein. RESULTS: AFP (20 mg/L) could promote the expression of FasL and TRAIL, and inhibit the expression of Fas and TRAILR of Bel7402 cells. For Jurkat cell line, AFP could suppress the expression of FasL and TRAIL, and stimulate the expression of Fas and TRAILR. AFP also could synergize with Bel7402 cells to inhibit the expression of FasL protein and TRAIL mRNA in Jurkat cells. The monoclonal antibody against AFP (anti-AFP) could abolish these functions of AFP. CONCLUSION: AFP is able to promote the expression of FasL and TRAIL in hepatoma cells and enhance the expression of Fas and TRAILR in lymphocytes. These could elicit the escape of hepatocellular carcinoma cells from the host's lymphocytes immune surveillance.

Alpha-fetoprotein stimulated the expression of some oncogenes in human hepatocellular carcinoma Bel 7402 cells.

AIM: To investigate the molecular mechanism of alpha-fetoprotein (AFP) on regulating the proliferation of human hepatocellular carcinoma cells. METHODS: Alpha-fetoprotein purified from human umbilical blood was added to cultured human hepatocellular carcinoma Bel 7402 cells in vitro for various treatment periods. The expression of c-fos, c-jun, and N-ras mRNA involved in proliferation and differentiation of cells was analyzed by Northern blot, and the expression of mutative p53 and p21(ras) proteins was determined by Western blot. RESULTS: The results showed that AFP (20 mg/L) stimulated mRNA expression of these oncogenes in Bel 7402 cells. The expression of c-fos mRNA increased by 51.1%, 60.9%, 96.0%, and 25.5% at 2, 6, 12, and 24 h, respectively. The expression of c-jun and N-ras mRNA reached to the maximum which increased by 81.3% and 59.9% as compared with the control after 6 h and 24 h incubation with AFP, respectively. Western blot assay also demonstrated that AFP promoted the expression of mutative p53 and p21(ras) proteins, and the increased rate of those proteins was 13.0%, 39.9%, and 70.9%, as well as 35.2%, 102.6%, and 46.8% at 6, 12, and 24 h, respectively, as compared with the control. Both human serum albumin (the same dosage as AFP) and monoclonal anti-AFP antibody failed to stimulate the expression of these oncogenes, but anti-AFP antibody could block the functions of AFP. CONCLUSION: The data indicate that AFP can stimulate the expression of some oncogenes to enhance the proliferation of human hepatocellular carcinoma Bel 7402 cells.

Enhancement of proliferation of HeLa cells by the alpha-fetoprotein.

A lot of investigations showed that alpha-fetoprotein(AFP) could regulate the proliferation of tumors or neoplastic cells. In order to study the enhancement of this function, AFP was extracted from human cord blood serum and was allowed to act on HeLa cell in vitro. The cellular proliferation was investigated by using the MTT assay and the [(3)H]-incorporation, and the cell cycle was analysed with flow cytometry. Radioactive immunosorbent assay was utilized to detect cAMP accumulation, protein kinase A activity and confocal microscopy was used f or scanning intracellular calcium. The expression of mutant p53 and p21(ras) protein were analyzed by Western blotting. Our results showed that AFP (>10 mg/L ) had strong effect of enhancement on the proliferation of HeLa cells; AFP (20 mg /L) could significantly increase the concentration of cAMP (300%), Ca(2+) (154.9%) and protein kinase A activity (100%). It was also shown that the expression of mutant p53 and p21(ras) protein of HeLa cells were enhanced after t he treatment with AFP (20 mg/L) for 24 hours. Compared with control, the expression of mutant p53 protein was increased by 81.1% (24 h), p21(ras) protein was increased by 96.2% (24 h) respectively; but the monoclonal antibody to AFP blocked these functions of AFP. In conclusion, AFP enhanced the proliferation of HeLa cells; AFP may regulate the growth of the cells by influencing the transmembrane signal pathway and enhancing the expression of the oncogenes.

The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells.

AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.

The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line.

AIM: The goal of this study was to characterize the AFP receptor, its possible signal transduction pathway and its proliferative functions in human hepatoma cell line Bel 7402. METHODS: Cell proliferation enhanced by AFP was detected by MTT assay, 3H-thymidine incorporation and S-stage percentage of cell cycle analysis. With radioactive labeled 125I-AFP for receptor binding assay; cAMP accumulation, protein kinase A activity were detected by radioactive immunosorbent assay and the change of intracellular free calcium (Ca2+i) was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncogenes N- ras, p 53, and p21( ras ) in the cultured cells in vitro were detected by Northern blotting and Western blotting respectively. RESULTS: It was demonstrated that AFP enhanced the proliferation of human hepatoma Bel 7402 cell in a dose dependent fashion as shown in MTT assay, (3)H-thymidine incorporation and S-phase percentage up to 2-fold. Two subtypes of AFP receptors were identified in the cells with Kds of 1.3 x 10(-9)mol.L(-1) and 9.9 x10(-8)mol. (-1)L respectively. Pretreatment of cells with AFP resulted in a significant increase (625%) in cAMP accumulation. The activity of protein kinase A activity were increased up to 37.5, 122.6, 73.7 and 61.2% at treatment time point 2, 6, 12 and 24 hours. The level of intracellular calcium were elevated after the treatment of alpha-fetoprotein and achieved to 204% at 4 min. The results also showed that AFP(20mg.L(-1)) could upregulate the expression of N- ras oncogenes and p 53 and p21( ras ) in Bel 7402 cells. In the later case,the alteration were 81.1%(12h) and 97.3%(12h) respectively compared with control. CONCLUSION: These results demonstrate that AFP is a potential growth factor to promote the proliferation of human hepatoma Bel 7402 cells. Its growth-regulatory effects are mediated by its specific plasma membrane receptors coupled with its transmembrane signaling transduction through the pathway of cAMP-PKA and intracellular calcium to regulate the expression of oncogenes.