Marsdenia tenacissima extract suppresses A549 cell migration through regulation of CCR5-CCL5 axis, Rho C, and phosphorylated FAK / 中国天然药物

Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP (an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.

Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Melanoma has the highest mortality among all superficial malignant tumors. The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets. As a molecular switch that controls tumor metastasis, Ras homology C (RhoC) has been correlated with tumor progression, especially tumor invasion and metastasis. However, little research has been done about the effects of RNA interference (RNAi) targeting RhoC on the invasion and metastasis of melanoma. In this study, we constructed an RNAi lentivirus vector targeting the RhoC gene of melanoma cells and identified its silencing effects on the RhoC gene.</p><p><b>METHODS</b>Based on the RhoC gene encoding information, three pGPU6/GFP/Neo-short hairpin (shRNA) plasmids were constructed. After detecting their silencing effects on the RhoC gene of A375 cells, the most effective pGPU6/GFP/Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453 targeting RhoC. The lentivirus vector was used to infect A375 cells, and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting.</p><p><b>RESULTS</b>The plasmids pGPU6/GFP/Neo-shRNA 336, pGPU6/GFP/Neo-shRNA 453, and pGPU6/GFP/Neo-shRNA 680 were constructed. After they were transfected into A375 cells, the expressions of RhoC mRNA and protein were 1.47 ± 0.26, 1.13 ± 0.16, 1.39 ± 0.11 and 70.98 ± 9.21, 50.67 ± 6.06, 65.77 ± 4.06, respectively. pGPU6/GFP/Neo-shRNA 453 was the most effective sequence, and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC. pLenti6.3-EGFP-453 was used to infect A375 cells. The expression of RhoC mRNA and protein were 1.05 ± 0.05 and 62.04 ± 15.86 in the lentivirus group, 4.21 ± 0.24 and 220.86 ± 24.07 in the negative lentivirus control group, and 4.63 ± 0.32 and 257.39 ± 12.30 in the normal control group respectively with the difference between the lentivirus group and the control groups being statistically significant (P < 0.05).</p><p><b>CONCLUSION</b>The successfully constructed pLenti6.3-EGFP-453 vector targeting the RhoC can effectively infect human melanoma A375 cells in vitro, and significantly inhibit the RhoC mRNA and protein expression.</p>

Effect of RhoC on hepatocellular carcinoma cell growth and related molecular mechanisms / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth.</p><p><b>METHODS</b>siRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup. Cell growth was assessed by MTT assay. AgNORs staining was applied to determine cell proliferation. Plate cell clone test was conducted to examine the capacity of cell clone formation. FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins. In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection.</p><p><b>RESULTS</b>The inhibition rate of RhoC was 82.3%. From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ± 0.10 vs. 0.73 ± 0.11 and 0.71 ± 0.07 respectively, P < 0.05). AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ± 0.35 vs. 3.47 ± 0.93 and 3.17 ± 0.78, P < 0.01). Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42)% vs. (70.58 ± 10.10)% and (69.83 ± 14.77)%, respectively, P < 0.01]. Cell cycle analysis by FACS showed that G(0)/G(1) cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ± 5.93)% vs. (57.05 ± 5.97)% and (52.99 ± 4.80)%, P < 0.05]. Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1(0.45 ± 0.21 vs. 1.25 ± 0.24 and 1.12 ± 0.15, respectively, P < 0.05)and CDK4 (0.55 ± 0.08 vs. 1.18 ± 0.32 and 1.10 ± 0.29, respectively, P < 0.05); the following genes were notably increased: p16(1.07 ± 0.23 vs. 0.36 ± 0.12 and 0.35 ± 0.13, respectively, P < 0.01)and p21(0.42 ± 0.12 vs. 0.17 ± 0.06 and 0.19 ± 0.08, respectively, P < 0.05). RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line. From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups (0.83 ± 0.10 vs. 0.54 ± 0.11 and 0.58 ± 0.55, respectively, P < 0.05).</p><p><b>CONCLUSIONS</b>RhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.</p>

Expression of RhoC and ROCK-1 and their effects on MAPK and Akt proteins in prostate carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect the expression of RhoC and Rho kinase 1 (ROCK-1) in prostate carcinoma, and explore the possible mechanism of RhoC/ROCK-1 in the pathogenesis of prostate carcinoma.</p><p><b>METHODS</b>Tissue specimens from 73 patients with prostate carcinoma and corresponding paracancerous tissues were obtained by prostate cancer biopsy or radical prostatectomy. The expression of RhoC/ROCK-1 mRNA was detected by RT-PCR. Western blot and immunohistochemistry were performed to dertect the expression of RhoC/ROCK-1 protein. Eukaryotic expression plasmids of RhoC were constructed and transfected into PC-3M-2B4 cells. p-MAPK and p-Akt were detected by Western bolt.</p><p><b>RESULTS</b>The expression levels of RhoC and ROCK-1 mRNA in the prostate carcinomas were significantly higher than those in corresponding paracancerous tissues [72.6% (53/73) vs. 34.2% (25/73); 68.5% (50/73) vs. 38.4% (28/73), P < 0.01], respectively. The results indicated that RhoC/ROCK-1 mRNA expression had no significant correlation with Gleason grade. However, the expression of RhoC/ROCK-1 mRNA showed a significant positive correlation with distant metastasis. The RhoC/ROCK-1 protein expression in prostate cancer was also higher than corresponding paracancerous tissues, and showed a significant positive correlation with p-MAPK and p-Akt expression levels. In addition, p-MAPK and p-Akt expression levels were up-regulated in the transcripts.</p><p><b>CONCLUSION</b>Expression levels of RhoC and ROCK-1 in prostate carcinoma are higher than those in corresponding paracancerous tissues, showing a significant positive correlation with distant metastasis. RhoC/ROCK-1 may be involved in the development, invasion and metastasis of prostate carcinoma.</p>

Expression and its relationship of PRL-3 and RhoC in non-small cell lung cancer / 中国肺癌杂志

<p><b>BACKGROUND AND OBJECTIVE</b>Phosphatase of regenerating liver-3 (PRL-3) is a newly identified protein-tyrosine phosphatase, which belongs to phosphatase of regenerating liver family, and plays a role in promoting tumor metastasis; Ras homologue C (RhoC) belongs to Rho subfamily of small-molecule G protein superfamily. However, the mechanisms of PRL-3 and RhoC are unknown. The aim of this study is to investigate the expressions of PRL-3 and RhoC proteins and their correlation to invasion and metastasis of non-small cell lung cancer (NSCLC), which may provide experiment evidence of the mechanism of PRL-3 in tumorigenesis and tumor-development.</p><p><b>METHODS</b>Immunohistochemical staining was used to detect the expressions of PRL-3 and RhoC in NSCLC in 92 cases, and statistical methods were used to analyse statistical significances of their expressions in different groups and their correlation.</p><p><b>RESULTS</b>The positive rates of PRL-3 and RhoC expressions in NSCLC were 69.6% (64/92) and 73.9% (68/92), respectively, and the expressions of PRL-3 and RhoC were closely correlated with TNM stage and lymphatic metastasis and pleural metastasis (P < 0.01), and they were correlated with each other (r = 0.754, P < 0.001).</p><p><b>CONCLUSION</b>The expressions of PRL-3 and RhoC are higher in the higher TNM stage and lymphatic metastasis and pleural metastasis cases, and closely correlate with each other in NSCLC, which suggests that PRL-3 and RhoC might be in the same signal pathway and PRL-3 might promote the distant metastasis of cancer cell by RhoC and downstream factors.</p>

Correlation of expression of RhoC with invasiveness of breast cancer cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of RhoC in breast cancer cells with different metastatic potential and its correlation with invasiveness.</p><p><b>METHODS</b>Expression of RhoC mRNA and protein in human breast cancer cells MCF-7 with low metastatic potential and MDA-MB-231 with high metastatic potential was detected by RT-PCR, Western blot, immunohistochemistry and immunofluorescence staining. Eukaryotic expression plasmids of RhoC were constructed and transfected into MCF-7 cells. The biological effects were observed, including in vitro invasion by Boyden charmber assay, motility by would healing assay, alteration of microfilament network by TRTIC-phalloidin staining and expression of p-Akt by Western blot assay.</p><p><b>RESULTS</b>The expression levels of RhoC mRNA and protein varied in the two different metastatic breast cancer cell lines. RhoC was significantly up-regulated in the highly metastatic cells in comparison to the weakly metastatic counterpart (P < 0.01). As shown by Boyden charmber assay, the invasive capacity of transfected cells overexpressing RhoC was significantly promoted as reflected by more penetrating cells (56.88 +/- 4.18) than that of the antisense transcripts (23.12 +/- 3.22), the negative (23.77 +/- 3.64) and blank controls (28.44 +/- 2.48). Further study by would healing assay indicated that cells overexpressing RhoC were more motile in actin-based active movement. The wound healing ratio after 24 h of the sense transcripts, antisense transcripts, negative controls and blank controls was 58.28% +/- 2.14%, 22.36% +/- 2.73%, 28.23% +/- 2.62%, 30.18% +/- 2.86%, respectively. The TRITC-phalloidin staining revealed less actin filament bundles and a reorganized cytoskeleton within the sense transcripts. In addition, p-Akt expression level was upregulated in the sense transcripts.</p><p><b>CONCLUSION</b>RhoC overexpression may promote the invasive capacity of human breast cancer cells in vitro and its expression level is positively correlated with the metastatic capacity of those cells. So RhoC may be a potential target in the development of a novel strategy for treating metastasis of breast cancer.</p>

Expressions of RhoC and osteopontin in esophageal squamous carcinoma and association with the patients' prognosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expressions of RhoC and osteopontin (OPN) protein in esophageal squamous carcinoma (ESC) and their association with the biological behavior of ESC.</p><p><b>METHODS</b>The expressions of RhoC and OPN protein were detected in 80 ESC cases by immunohistochemistry.</p><p><b>RESULTS</b>The positive expression rate of RhoC was 66.25% in these ESC cases. The rate was significantly higher in cases with lymph node metastasis than in those without (r(s)=-2.115, P<0.05), but RhoC expression was not associated with the tumor diameter, differentiation or TNM grade (P>0.05). The RhoC-positive patients had significantly shorter survival time than the negative patients (P<0.001). All the 80 ESC patients were positive for OPN expression, and OPN expression levels were correlated with the differentiation (chi(2)=10.766, P<0.05) and lymph node metastasis of the tumor (r(s)=-2.289, P<0.05), but not with the tumor diameter or TNM grade (P>0.05). Higher expression level of OPN was closely related to shorter survival time of the patients (P<0.05). A positive correlation was found between RhoC protein and OPN expressions (r(s)= 0.408, P<0.001) in these cases.</p><p><b>CONCLUSION</b>The expressions of RhoC and OPN protein are closely related to lymph node metastasis of ESC and the patients survival time, and therefore may serve the purpose of prognostic evaluation of ESC.</p>

Expression of RhoC protein in human primary hepatocellular carcinoma and its clinical significance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To detect the expression of RhoC protein in human primary hepatocellular carcinoma (HCC) and pericancerous liver (PCL) tissues and its relation to HCC prognosis.</p><p><b>METHODS</b>Immunohistochemical method was used to detect the expression of RhoC protein in HCC, PCL of 43 patients. Thirty-six patients were followed up after they received radical resection.</p><p><b>RESULT</b>The expression of RhoC protein in HCC was significantly higher than that in PCL. Rhoc expression was increased in cases with poor differentiation, portal vascular invasion, unintact envelope and multiple masses. The survival analysis showed that HCC with lower RhoC protein expression had better clinical outcomes.</p><p><b>CONCLUSION</b>RhoC protein expression is correlated with infiltration and metastasis in HCC. RhoC protein can be used as a prognostic indicator for HCC.</p>

Expressions of RhoC, CD44v6 and ICAM-1 in human gastric cancer and its clinicopathological significance / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expressions of RhoC, CD44v6 and ICAM-1 in gastric cancer and their correlations.</p><p><b>METHODS</b>SABC immunohistochemistry was used to detect the expressions of RhoC, CD44v6 and ICAM-1 in the specimens from 40 cases with gastric cancer. Their correlations were reviewed by clinicopathological data.</p><p><b>RESULTS</b>The expression of RhoC, CD44v6 and ICAM-1 were not correlated with tumor differentiation and invasion depth (P> 0.05), but significantly correlated with lymph metastasis and pTNM stage (P< 0.05). There was a significant correlation between the expression of RhoC and the expression of CD44v6 or ICAM-1 (r=0.355, P=0.006; r=0.354, P=0.003) respectively. If RhoC was positive-expressed, and either of CD44v6 or ICAM-1 was positive-expressed, the sensitivity and the specificity for predicting the lymphatic metastasis was 93.75%, 62.5% respectively.</p><p><b>CONCLUSIONS</b>The positive expressions of RhoC, CD44v6 and ICAM-1 are useful biological markers for predicting the metastatic potential of gastric cancer. The combined detection of three markers is a useful method for predicting lymphatic metastasis of gastric cancer.</p>

Overexpression of the RhoC gene correlates with invasion and metastasis of hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>Rho, a ras homologous gene, encodes a group of GTP-binding proteins. Our previous study suggested that one member of the Rho gene family, RhoC, was related to the progression of human hepatocellular carcinoma (HCC). This study is to elucidate correlation of Rho overexpression with invasion and metastasis of HCC.</p><p><b>METHODS</b>The expression level of RhoC mRNA and protein in 25 cases of HCC and adjacent non-cancerous liver tissue was examined by RT-PCR and Western blot. Mutation of RhoC gene was examined by PCR-SSCP.</p><p><b>RESULTS</b>The expression of RhoC mRNA and protein was found in all HCC and adjacent non-cancerous liver tissue. The expression level of RhoC mRNA and protein was significantly higher in tumor tissue than in adjacent non-cancerous liver tissue (1.8 +/- 1.1 vs 1.0 +/- 0.7; 33 992 +/- 10 384 vs 17 342 +/- 9998, P < 0.01). The degree of RhoC overexpression was even more marked in metastatic lesions than in primary tumors (P < 0.01). Overexpression of the rhoC gene was significantly correlated with such clinic-pathological findings as cell differentiation, portal vein invasion, number of primary tumor nodules and metastatic lesions (P < 0.05). Mutation of RhoC gene was found in none of the HCC specimens examined.</p><p><b>CONCLUSION</b>Overexpression of RhoC gene may play an important role in carcinogenesis and progression of HCC.</p>

Correlation of expression of RhoA (RhoC and their effector ROCK-1 with malignant phenotype of ovarian cancer cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of RhoA, RhoC and their effector ROCK-1 in four ovarian cancer cell lines in vitro and their correlation with invasiveness.</p><p><b>METHODS</b>Expression of RhoA, RhoC and ROCK-1 mRNA and protein in four ovarian cancer cell lines SW626, Skov-3, A2780 and Caov-3 was detected by RT-PCR and Western blot assay. Invasion assay was done in Boyden chamber.</p><p><b>RESULTS</b>The expression levels of RhoA, RhoC and ROCK-1 mRNA and protein varied in the four different cell lines examined. The expression level of RhoC, but not RhoA and ROCK-1, was significantly correlated with the invasive capability of these cells in vitro (r = 0.95, P < 0.01). Expression of RhoA at the level of transcription was not correlated with that at the translation level. The expression of RhoA and RhoC did not correlate with that of ROCK-1.</p><p><b>CONCLUSION</b>Expression level of RhoC may serve as an independent parameter in evaluating metastasis and become a new target in inhibiting ovarian cancer metastasis.</p>