The purpose of this study was to explore the effect and mechanism of Xihuang Pills on rats with precancerous lesions of the breast. Of 48 healthy female rats, 8 were randomly selected as blank group, and the other 40 were treated with 7,12-dimethylbenzanthracene(DMBA) combined with estrogen and progestin to establish a model of precancerous lesions of the breast. The successfully modeled rats were randomly divided into a model group, a tamoxifen group(1.8 mg·kg~(-1)·d~(-1)), a Xihuang Pills low-dose group(0.3 g·kg~(-1)·d~(-1)), a medium-dose group(0.6 g·kg~(-1)·d~(-1)) and a high-dose group(1.2 g·kg~(-1)·d~(-1)). After 30 days of admi-nistration, the histopathological changes of viscera and breast were observed by haematoxylin and eosin(HE) staining, and the visceral index was calculated. Enzyme linked immunosorbent assay(ELISA) was used to detect the contents of estradiol(E_2) and progesterone(P) in serum. The protein expressions of vascular endothelial growth factor(VEGF) and fibroblast growth factor 2(FGF2) were detected by immunohistochemistry. The protein expressions of VEGF, vascular endothelial growth factor receptor 2(VEGFR2), phosphorylated-vascular endothelial growth factor receptor 2(p-VEGFR2), B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were detected by Western blot and the mRNA expressions of VEGF, FGF2, CXC-chemokine receptor 4(CXCR4), cysteine aspartic acid-specific protease(caspase-3), and stromal cell-derived factor 1(SDF-1) were detected by real-time polymerase chain reaction(RT-PCR). HE staining revealed that the model group had some liver and kidney damages and severe hyperplastic mammary tissue, while the Xihuang Pills high-dose group had mild hyperplasia. Compared with the model group, the Xihuang Pills groups had lo-wer ovarian coefficient(P<0.05 or P<0.01) and Xihuang Pills high-dose group had lower uterine coefficient(P<0.01). ELISA results showed that compared with the model group, expressions of E_2 and P in Xihuang Pills high-dose group were significantly decreased(P<0.05 or P<0.01). Immunohistochemistry, Western blot and RT-PCR indicated that compared with the conditions in the model group, the protein and mRNA expressions of VEGF and FGF2 in the Xihuang Pills groups were down-regulated(P<0.05 or P<0.01), and the protein expression of Bcl-2 was lowered(P<0.01); there was a decrease in the protein expressions of VEGFR2 and p-VEGFR2(P<0.01), a down-regulation in the mRNA expressions of CXCR4 and SDF-1(P<0.01), while an increase in the mRNA expression of caspase-3(P<0.01) in both Xihuang Pills medium-dose and high-dose groups; the protein expression of Bax in Xihuang Pills high-dose group was increased(P<0.01). The above results indicated that Xihuang Pills can effectively intervene in precance-rous lesions of the breast, and the mechanism may be related to the regulation of E_2 and P secretion as well as the inhibition of angiogenesis and chemokine receptor expression, thus controlling the occurrence of precancerous lesions of the breast in rats.
Rats ; Female ; Animals ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; Vascular Endothelial Growth Factor A/metabolism* ; Caspase 3 ; Vascular Endothelial Growth Factor Receptor-2 ; Fibroblast Growth Factor 2 ; Proto-Oncogene Proteins c-bcl-2 ; 9,10-Dimethyl-1,2-benzanthracene/toxicity* ; Precancerous Conditions ; Hyperplasia ; Receptors, Chemokine ; RNA, Messenger

The study aimed to investigate the intervening role of Didang decoction (DDD) at different times in macrovascular endothelial defense function, focusing on its effects on the AMP-activated protein kinase (AMPK) signaling pathway. The effects of DDD on mitochondrial energy metabolism were also investigated in rat aortic endothelial cells (RAECs). Type 2 diabetes were induced in rats by streptozotocin (STZ) combined with high fat diet. Rats were randomly divided into non-intervention group, metformin group, simvastatin group, and early-, middle-, late-stage DDD groups. Normal rats were used as control. All the rats received 12 weeks of intervention or control treatment. Western blots were used to detect the expression of AMP-activated protein kinase α1 (AMPKα1) and peroxisome proliferator-activated receptor 1α (PGC-1α). Changes in the intracellular AMP and ATP levels were detected with ELISA. Real-time-PCR was used to detect the mRNA level of caspase-3, endothelial nitric oxide synthase (eNOS), and Bcl-2. Compared to the diabetic non-intervention group, a significant increase in the expression of AMPKα1 and PGC-1α were observed in the early-stage, middle-stage DDD groups and simvastatin group (P < 0.05). The levels of Bcl-2, eNOS, and ATP were significantly increased (P < 0.05), while the level of AMP and caspase-3 were decreased (P < 0.05) in the early-stage DDD group and simvastatin group. Early intervention with DDD enhances mitochondrial energy metabolism by regulating the AMPK signaling pathway and therefore may play a role in strengthening the defense function of large vascular endothelial cells and postpone the development of macrovascular diseases in diabetes.
AMP-Activated Protein Kinases ; metabolism ; Adenosine Triphosphate ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; Cardiovascular Diseases ; metabolism ; prevention & control ; Caspase 3 ; metabolism ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; metabolism ; Diabetes Mellitus, Type 2 ; complications ; drug therapy ; metabolism ; Diptera ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; drug effects ; metabolism ; Energy Metabolism ; drug effects ; Leeches ; Mitochondria ; drug effects ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; metabolism ; Phytotherapy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Prunus persica ; Rats, Sprague-Dawley ; Rheum ; Signal Transduction

OBJECTIVEThis study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish.

METHODSThe in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels.

RESULTSR1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs.

CONCLUSIONR1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.

Animals ; Blood Vessels ; pathology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Collagen ; pharmacology ; Disease Models, Animal ; Drug Combinations ; Ginsenosides ; chemistry ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; enzymology ; physiology ; Humans ; Laminin ; pharmacology ; Neovascularization, Physiologic ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Proteoglycans ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Zebrafish

Marsdeniae tenacissimae extract (MTE), commonly known as Xiao-Ai-Ping in China, is a traditional Chinese herb medicine capable of inhibiting proliferation and metastasis and boosting apoptosis in various cancer cells. However, little is known about the contribution of MTE towards tumor angiogenesis and the underlying mechanism. The present study aimed to evaluate the effects of MTE on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) and the molecular mechanism. 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt (MTS) and PI-stained flow cytometry assays revealed that MTE dose-dependently reduced the proliferation of HUVECs by arresting cell cycle at S phase (P < 0.05). Annexin V-FITC/PI-stained flow cytometry confirmed that MTE (160 μL·L) enhanced the apoptosis of HUVECs significantly (P < 0.001). Real-time quantitative RT-PCR and Western blot analyses showed an increase in Bax expression and a sharply decline in Bcl-2 expression; caspase-3 was activated simultaneously in a dose-dependent manner (P < 0.05). Further study observed the dose-dependent down-regulation of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2), P2Y6 receptor (P2Y6R), and chemokine (C-C motif) ligand 2 (CCL-2), along with the activation of PKC Δ and up-regulation of p53 in a dose-dependent manner in MTE-treated selected cells (P < 0.05). Collectively, the results from the present study suggested that MTE suppressed the proliferation by attenuating CCL-2-mediated VEGF/VEGFR2 interactions and promoted the apoptosis through PKCΔ-induced p53-dependent mitochondrial pathway in HUVECs, supporting that MTE may be developed as a potent anti-cancer medicine.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Marsdenia ; chemistry ; Plant Extracts ; pharmacology ; Protein Kinase C ; genetics ; metabolism ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

This study was aimed to investigate the mRNA and protein expression of CTGF, CYR61, VEGF-C and VEGFR-2 in bone marrow of patients with leukemia, and to analyze the role and clinical significance of these 4 factors in genesis and development of leukemia, infiltration and metastasis of leukemic cells. A total of 100 cases of newly diagnosed leukemia, 26 cases of acute leukemia in complete remission and 30 controls were enrolled in this study. The mononuclear cells of bone marrow were collected, the mRNA and protein expression levels of CTGF, CYR61, VEGF-C, VEGFR-2 in leukemia patients and controls were detected by real time PCR and Western blot, respectively. The results showed that the mRNA and protein expression levels of above mentioned 4 factors were significantly higher than those in control (P < 0.05), only CTGF mRNA expression in AL patients after complete remission showed statistical difference as compared with control (P < 0.05), but the expression of CTGF mRNA showed statistical significance in different bone marrow hyperplasia of acute leukemia (P < 0.05). The expression level of CTGF protein showed difference in different chromosome karyotypes of leukemia (P < 0.05). The expression levels of CYR61 and VEGF-C proteins showed statistical difference in different bone marrow hyperplasia of acute leukemia (P < 0.05). The expression level of CTGF, CYR61, VEGF-C mRNA and protein in CML group were higher than that in control group. The expression levels of CTGF and CYR61 protein were higher than that in control. The mRNA and protein expression levels of above-mentioned 4 factors in sex and infiltration lf leukemic cells did not show statistical significance(P < 0.05). In correlative analysis, the mRNA expressions of above mentioned 4 factors were positively correlated with bone marrow blast count(P < 0.05), the protein expression of CTGF, CYR61 and VEGF-C were positively correlated with bone marrow blast count. It is concluded that the CTGF, CYR61, VEGF-C and VEGFR-2 mRNA and protein play a role in acute leukemia. In acute leukemia (AML/ALL), the expression of above mentioned factor was high, but except VEGFR-2. Most of them were positively correlated with bone marrow blast count. Joint block of these angiogenesis-related factors is likely to play an important role in targeting treatment of leukemia.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; metabolism ; pathology ; Case-Control Studies ; Child ; Child, Preschool ; Connective Tissue Growth Factor ; metabolism ; Cysteine-Rich Protein 61 ; metabolism ; Female ; Humans ; Leukemia ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Vascular Endothelial Growth Factor C ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Young Adult

OBJECTIVETo study the phenotype and tumorigenicity of SHG-44 glioma stem cell spheres and the pathological characteristics of their xenograft tumors.

METHODSSHG-44 glioma cells were cultured under neural stem cell medium and glioma stem cell spheres were collected. Immunocytochemistry was used to dectet the expression of CD133, nestin, A2B5, vimentin, VEGFR-2 and IDH R132H. Cell spheres were induced using serum-containing medium, and the expression of CD133, nestin, vimentin, GFAP, β-III tubulin and GalC in the cell spheres were detected. The expression of CD133, nestin, VEGFR-2, GFAP, S-100 and CD34 in the intracranial xenograft tumor tissues was detected using immunohistochemistry. The pathological characteristics of orthotopic xenograft tumors generated from the SHG-44 glioma cells and SHG-44 glioma stem cell spheres were compared.

RESULTSSHG-44 glioma stem cell spheres were collected successfully after cultured under neural stem cell medium. The ratio of CD133(+) cells in the passage 10 SHG-44 glioma stem cell spheres was (71.63 ± 5.92)%, significantly higher than that in the SHG-44 glioma cells [(1.95 ± 1.45)%]. Immunocytochemistry showed that in the SHG-44 glioma cell spheres, the ratio of nestin(+) cells was (84.06 ± 7.58)%, vimentin(+) cells (29.11 ± 3.44)%, VEGFR 2(+) cells (64.44 ± 3.69)%, and A2B5(+) cells (14.08 ± 2.19)%. A subpopulation of cells with mutation of IDH R132H was detected harboring in the SHG-44 glioma cell spheres. After induction of differentiation with serum-containing medium, the ratio of CD133(+) cells was (1.89 ± 1.27)%, nestin(+) cells (6.67 ± 2.75)%, vimentin(+) cells (93.75 ± 2.95)%, GFAP (+) cells (91.33 ± 4.75)%, β-III tubulin(+) cells (82.36 ± 4.02)%, and GalC(+) cells (8.92 ± 3.19)%. Immunohistochemistry showed positive expression of GFAP, S-100, VEGFR-2, and negative of CD133 and nestin in the orthotopic xenograft tumors. A very small amount of human-specific CD34 cells formed a tubular structure. Compared with the SHG-44 glioma cell-formed xenograft tumor, the SHG-44 glioma stem cell-formed xenograft tumor exhibited a higher local invasiveness.

CONCLUSIONSSHG-44 glioma cell spheres are successfully collected after cultured under neural stem cell medium. They belong to the CD133(+)A2B5(-) GSC subpopulation, highly expressing VEGFR-2, possess the ability of both self-renewal and multi-directional differentiation, and may participate in the formation of vasculogenic mimicry.

Animals ; Brain Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Glial Fibrillary Acidic Protein ; metabolism ; Glioma ; metabolism ; pathology ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Neoplastic Stem Cells ; metabolism ; pathology ; S100 Proteins ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

endothelial growth factor (VEGF)-C and its tyrosine kinase receptor, VEGF receptor (VEGFR)-3 are recently known to have lymphangiogenic activities in various tumor types. In this study, we determined whether the expression of lymphangiogenic factors correlate with nodal metastasis or survival in a nude mouse model of oral squamous cell carcinoma (OSCC).METHODS: Three OSCC cells (KB, SCC4, SCC9) were xenografted into the right mandibular gland of athymic nude mice. The mice were followed for tumor development and growth, and the mice were sacrificed when they had lost more than 20% of their initial body weight, or the diameter of the induced tumor exceeds 20 mm. After necropsy, the murine tumors were examined histologically and radiologically (micro-positron emission tomography computed tomography) for regional or distant metastasis. We performed immunohistochemical assays with anti-VEGF-C, VEGFR-3, CD105, and D2-40 antibodies. Immunofluorescence double staining for LYVE-1/CD31 was also performed. To quantify the VEGF-C and VEGFR-3 level in the cancer tissue, Western blotting was performed. Finally, we determined the correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time.RESULTS: OSCC tumor cells into the mandibular gland of the nude mice successfully resulted in the formation of recapitulating orthotopic tumor. Tumor cells of the induced tumor did not express VEGF-C. VEGF-C/VEGFR-3 expression was mainly distributed in the endothelial cells of the stromal area. There were no correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time of mice injected with different OSCC cell lines.CONCLUSION: An recapitulating orthotopic model of OSCC in nude mice was established, which copies the cervical nodal metastasis of human OSCC. Overexpression of lymphangiogenic factors seems to have no effect on survival of hosts in this in vivo experiment.]]>
Animals ; Antibodies ; Blotting, Western ; Body Weight ; Carcinoma, Squamous Cell ; Coat Protein Complex I ; Endothelial Cells ; Fluorescent Antibody Technique ; Growth and Development ; Humans ; Mice ; Mice, Nude ; Neoplasm Metastasis ; Protein-Tyrosine Kinases ; Receptors, Vascular Endothelial Growth Factor ; Survival Rate ; Transplantation, Heterologous ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factor C ; Vascular Endothelial Growth Factor Receptor-3

OBJECTIVETo observe the effect of C-reactive protein (CRP) on the expressions of Notch pathway components in human peripheral blood endothelial progenitor cells (EPC) in vitro.

METHODSMononuclear cells isolated by density gradient centrifugation of human peripheral blood mixed with 6% hydroxyethyl starch (Hes) were plated on fibronectin-coated 6-well culture dishes. After 7 days, the adherent cells were cultured in the presence of 10 and 20 mg/L CRP for 48 h, and the proliferation, migration, and adhesion abilities of the cells were observed. The mRNA expressions of Notch-1 and its ligand Jagged-1 in the EPCs were measured by RT-PCR, and their protein expressions by Western blotting.

RESULTSCRP at 10 and 20 mg/L caused a significant reduction in the number of viable EPCs (61∓3 and 54∓3, respectively) as compared with PBS (71∓4, P<0.05). CRP also resulted in a significant suppression of the proliferation, migration and adhesion capacities of the EPCs. The mRNA and protein expressions of Jagged-1 and Notch-1 in the EPCs significantly increased following CRP exposure in comparison with PBS treatment.

CONCLUSIONCRP can suppress the proliferation, migration and adhesion capacities of the EPCs probably by affecting the expressions of the Notch-1 pathway components.

C-Reactive Protein ; pharmacology ; Calcium-Binding Proteins ; genetics ; metabolism ; Cell Adhesion ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Jagged-1 Protein ; Leukocytes, Mononuclear ; cytology ; metabolism ; Membrane Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptor, Notch1 ; genetics ; metabolism ; Serrate-Jagged Proteins ; Signal Transduction ; drug effects ; Stem Cells ; cytology ; metabolism

OBJECTIVETo explore the effect of HMGB1 on the VEGF-C expression and proliferation of esophageal squamous cancer cells as well as its possible mechanism.

METHODSA cassette encoding siRNA targeting HMGB1 mediated by rAAV was constructed, the rAAV-siHMGB1-hrGFP, and a vector encoding siRNA mismatching HMGB1 was constructed, the rAAV-miHMGB1-hrGFP. This experiment in vitro included three groups, namely, the blank control group (group A) of KYSE150 cells transfected by rAAV-hrGFP, negative mismatch control group (group B) of KYSE150 cells transfected with rAAV-miHMGB1-hrGFP, and RNA interference group (group C) of KYSE150 cells transfected with rAAV-siHMGB1-hrGFP. We examined the expression of HMGB1 mRNA and protein in the three group cells by real-time PCR and Western blot after 24 h and 48 h, respectively. Then, VEGF-C expression and cell proliferation in the three group cells with or without sRAGE, as an inhibitor of RAGE signal pathway, were assayed by ELISA and MTT after 24 h.

RESULTSThe expression of HMGB1 mRNA and protein in KYSE150 cells in vitro in the group C transfected with rAAV-siHMGB1-hrGFP at the final concentration of 2×10(6) v.g/cell was significantly lower than that of the group A or B after 24 h and 48 h (P < 0.01). The VEGF-C expression of KYSE150 cells was (502.43 ± 13.10) pg/ml in the group C, significantly reduced in comparison with that of the group A (686.40 ± 10.94) pg/ml or group B (682.31 ± 9.61) pg/ml after 24 h (P < 0.05). At the same time, the proliferation of KYSE150 cells in the group C was significantly inhibited compared with that of groups A and B after 24 h (P < 0.01). Moreover, sRAGE at the final concentration of 0.2 µg/ml inhibited the VEGF-C expression and proliferation of KYSE150 cells compared with the corresponding group without sRAGE after 24 h (P < 0.01 or P < 0.05). However, there was no significant difference of the VEGF-C expression and proliferation of KYSE150 cells with sRAGE in the group C compared with that of cells with sRAGE of the group A or group B after 24 h (P > 0.05).

CONCLUSIONSIn esophageal squamous cell carcinoma, HMGB1 can promote the VEGF-C expression and proliferation of the cancer cells through RAGE signal pathway, and HMGB1-RAGE may become a potential target for cell proliferation and lymph node metastasis of this cancer.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Dependovirus ; genetics ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; HMGB1 Protein ; biosynthesis ; genetics ; Humans ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Signal Transduction ; Transfection ; Vascular Endothelial Growth Factor C ; metabolism

BACKGROUNDTwist is a highly conserved epithelial-mesenchymal transcription factor that has been reported to be a key factor in tumor malignancy, including lymph node metastasis. It represents the major step of dissemination and serves as a chief prognostic indicator of disease progression. However, the mechanism by which Twist regulates lymph node metastasis remains incompletely understood. Studies on the mechanism of metastasis are thus required for determining appropriate therapeutic strategies.

METHODSImmunohistochemistry for lymphatic vessel endothelial receptor 1 (LYVE-1), Ki-67, Twist, vascular endothelial growth factor C (VEGF-C), and vascular endothelial growth factor receptor 3 (VEGFR-3) was performed to detect lymphatic vessel density (LVD), cell proliferation levels and the expressions of Twist, VEGF-C, and VEGFR-3 were determined from 66 primary supraglottic carcinoma tissue samples from 36 patients with lymph node metastasis (pathological N+, pN+) and 30 patients without metastasis (pathological N0, pN0). Western blotting analysis of the proteins in pN+ and pN0 primary tumors was used to characterize the expressions of Twist, VEGF-C, and VEGFR-3 further.

RESULTSThe LVD was 22.4 ± 10.3 in pN+ patients and 6.8 ± 4.1 in pN0 ones. For Ki-67, the number of proliferous cells in pN+ patients was greater than that in pN0 ones. Both, however, were associated with their clinical nodal stages. In pN+ patients, Twist, VEGF-C, and VEGFR-3 expressions were 86.11% (31/36), 80.56% (29/36), and 58.33% (21/36), respectively. These values were higher than those found for pN0 patients (i.e., 13/30, 11/30, and 7/30, respectively) (P < 0.05). Among the samples with Twist expression, 88.64% were VEGF-C-positive and 59.09% were VEGFR-3-positive. The pN0 counterparts were 4.55% and 9.09%, respectively (P < 0.05). The expressions of Twist, VEGF-C, and VEGFR-3 in pN+ patients obtained through Western blotting analysis were significantly higher than those in pN0 patients, and the levels of VEGF-C and VEGFR-3 were positively correlated with that of Twist.

CONCLUSIONSTwist expression correlates with lymph node metastasis. The mechanism involved in such a correlation may be related to lymphangiogenesis.

Adult ; Aged ; Blotting, Western ; Female ; Humans ; Immunohistochemistry ; Laryngeal Neoplasms ; complications ; metabolism ; Lymphangiogenesis ; genetics ; physiology ; Lymphatic Metastasis ; genetics ; pathology ; Male ; Middle Aged ; Twist-Related Protein 1 ; genetics ; metabolism ; Vascular Endothelial Growth Factor C ; metabolism ; Vascular Endothelial Growth Factor Receptor-3 ; metabolism