A new method for predicting the prognosis of colorectal cancer patients through a combination of multiple tumor-associated macrophage markers at the invasive front.

Tumor-associated macrophages (TAMs) are closely related to tumorigenesis and metastasis of multiple cancer types. The infiltration of TAMs is used for predicting the prognosis of cancers, including colorectal cancer (CRC). However, the density and prognostic significance of M1 and M2 TAM phenotypes in the intratumor versus the invasive front (IF) are largely unknown in CRC. In this study, CD68 was selected as a general marker of TAMs, CD11c, NOS2 and CXCL10 as markers for M1 phenotype and CD163, CD206, CD115 as markers for M2 phenotype. Firstly, immunohistochemistry staining and double-labeling immunofluorescence staining showed that M1 molecular markers (NOS2, CXCL10, CD11c) were lowly expressed at both IF and intratumor, while M2 molecular markers (CD163, CD206, CD115) were highly expressed mainly at IF. Moreover, we also demonstrated that three M1 molecular markers including NOS2, CXCL10 and CD11c were correlated to each other. Meanwhile, three M2 molecular markers including CD163, CD206, and CD115 were also correlated to each other. Patients with low expression of three M1 molecular markers (NOS2/CXCL10/CD11c) exhibited low overall survival (OS) rate, whereas patients with high expression of three M2 molecular markers (CD163/CD206/CD115) exhibited low OS rate. We also observed that the prognostic value of treble markers combination (NOS2/CXCL10/CD11c or CD163/CD206/CD115) was superior to that of single marker. Together, our results reveal the combination of treble TAMs markers (NOS2/CXCL10/CD11c or CD163/CD206/CD115) could better evaluate the prognosis of CRC patients, which might be used as a more comprehensive method for predicting the prognosis of CRC patients.

Tumor bud-derived CCL5 recruits fibroblasts and promotes colorectal cancer progression via CCR5-SLC25A24 signaling.

BACKGROUND: Tumor budding is included in the routine diagnosis of colorectal cancer (CRC) and is considered a tumor prognostic factor independent of TNM staging. This study aimed to identify the fibroblast-mediated effect of tumor bud-derived C-C chemokine ligand 5 (CCL5) on the tumor microenvironment (TME). METHODS: Recruitment assays and a human cytokine array were used to detect the main cytokines that CRC tumor buds secrete to recruit fibroblasts. siRNA transfection and inhibitor treatment were used to investigate the role of fibroblast CCL5 receptors in fibroblast recruitment. Subsequently, transcriptome sequencing was performed to explore the molecular changes occurring in fibroblasts upon stimulation with CCL5. Finally, clinical specimens and orthotopic xenograft mouse models were studied to explore the contribution of CCL5 to angiogenesis and collagen synthesis. RESULTS: Hematoxylin-eosin staining and immunochemistry revealed a higher number of fibroblasts at the invasive front of CRC tissue showing tumor budding than at sites without tumor budding. In vitro experiments demonstrated that CCL5 derived from tumor buds could recruit fibroblasts by acting on the CCR5 receptors on fibroblasts. Tumor bud-derived CCL5 could also positively regulate solute carrier family 25 member 24 (SLC25A24) expression in fibroblasts, potentially activating pAkt-pmTOR signaling. Moreover, CCL5 could increase the number of α-SMAhigh CD90high FAPlow fibroblasts and thus promote tumor angiogenesis by enhancing VEGFA expression and making fibroblasts transdifferentiate into vascular endothelial cells. Finally, the results also showed that CCL5 could promote collagen synthesis through fibroblasts, thus contributing to tumor progression. CONCLUSIONS: At the invasive front of CRC, tumor bud-derived CCL5 can recruit fibroblasts via CCR5-SLC25A24 signaling, further promoting angiogenesis and collagen synthesis via recruited fibroblasts, and eventually create a tumor-promoting microenvironment. Therefore, CCL5 may serve as a potential diagnostic marker and therapeutic target for tumor budding in CRC.

MYH9-dependent polarization of ATG9B promotes colorectal cancer metastasis by accelerating focal adhesion assembly.

Tumour metastasis is a major reason accounting for the poor prognosis of colorectal cancer (CRC), and the discovery of targets in the primary tumours that can predict the risk of CRC metastasis is now urgently needed. In this study, we identified autophagy-related protein 9B (ATG9B) as a key potential target gene for CRC metastasis. High expression of ATG9B in tumour significantly increased the risk of metastasis and poor prognosis of CRC. Mechanistically, we further find that ATG9B promoted CRC invasion mainly through autophagy-independent manner. MYH9 is the pivotal interacting protein for ATG9B functioning, which directly binds to cytoplasmic peptide segments aa368-411 of ATG9B by its head domain. Furthermore, the combination of ATG9B and MYH9 enhance the stability of each other by decreasing their binding to E3 ubiquitin ligase STUB1, therefore preventing them from ubiquitin-mediated degradation, which further amplified the effect of ATG9B and MYH9 in CRC cells. During CRC cell invasion, ATG9B is transported to the cell edge with the assistance of MYH9 and accelerates focal adhesion (FA) assembly through mediating the interaction of endocytosed integrin ß1 and Talin-1, which facilitated to integrin ß1 activation. Clinically, upregulated expression of ATG9B in human CRC tissue is always accompanied with highly elevated expression of MYH9 and associated with advanced CRC stage and poor prognosis. Taken together, this study highlighted the important role of ATG9B in CRC metastasis by promoting focal adhesion assembly, and ATG9B together with MYH9 can provide a pair of potential therapeutic targets for preventing CRC progression.

Role of TGFß3-Smads-Sp1 axis in DcR3-mediated immune escape of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of tumour-associated mortality worldwide, but no significant improvement in treating HCC has been reported with currently available systemic therapies. Immunotherapy represents a new frontier in tumour therapy. Therefore, the immunobiology of hepatocarcinoma has been under intensive investigation. Decoy receptor 3 (DcR3), a member of the tumour necrosis factor receptor (TNFR) superfamily, is an immune suppressor associated with tumourigenesis and cancer metastasis. However, little is known about the role of DcR3 in the immunobiology of hepatocarcinoma. In this study, we found that overexpression of DcR3 in HCC is mediated by the TGFß3-Smad-Sp1 signalling pathway, which directly targets DcR3 promoter regions. Moreover, overexpression of DcR3 in HCC tissues is associated with tumour invasion and metastasis and significantly promotes the differentiation and secretion of Th2 and Treg cells while inhibiting the differentiation and secretion of Th1 cells. Conversely, knockdown of DcR3 expression in HCC significantly restored the immunity of CD4+ T cells. Inhibition of DcR3 expression may provide a novel immunotherapeutic approach to restoring immunity in HCC patients.

Cancer-associated fibroblasts promote colorectal cancer progression by secreting CLEC3B.

Nontumour cells in the tumour microenvironment, especially fibroblasts, contribute to tumour progression and metastasis. The occurrence and evolution of colorectal cancer (CRC) is closely related to cancer-associated fibroblasts (CAFs). The aim of this work was to evaluate the effects of the growth factors and cytokines secreted by CAFs on CRC progression. The secreted cytokines were examined in CAFs by Human Cytokine Antibody array. We screened 37 differentially secreted cytokines in the culture supernatants of CAFs and NFs. CLEC3B, attractin, kallikrein 5 and legumain were selected for further verification. CLEC3B was more highly expressed in the stroma of CRC tissues than the other 3 cytokines. Immunohistochemistry revealed that CLEC3B expression was associated with serosal invasion by CRC. Patients with co-expression of CLEC3B and α-SMA had worse survival outcomes than those with only CLEC3B or α-SMA expression. CLEC3B secreted from CAFs may promote tumour migration. Knockdown of endogenous CLEC3B in CAFs markedly decreased CRC cell migration, while recombinant human CLEC3B clearly promoted CRC cell migration and actin remodelling. In conclusion, our findings suggest that CAFs promote the CRC cell migration and skeletal reorganization by secreting CLEC3B. CLEC3B might be a potential therapeutic molecule for CRC treatment.

Gastric aggressive fibromatosis: report of a case and review of the literature.

OBJECTIVES: To describe a rare case of aggressive fibromatosis of the stomach and discuss the differential diagnoses. METHODS: A 47-year-old man presented with nonspecific abdominal pain. Gastroscopy revealed stomach wall swelling. An antral gastrectomy was performed. Histological examination revealed spindle-shaped cells and morphology typical of aggressive fibromatosis. We performed a literature search to identify conditions with features similar to those of aggressive fibromatosis. RESULTS: Aggressive fibromatosis does not metastasize, but it is locally invasive and has a tendency to relapse; however, our patient has not had recurrence > 1 year after surgery. Aggressive fibromatosis of the stomach may be confused with an inflammatory fibroid polyp, a gastrointestinal stromal tumor, schwannoma, leiomyoma, inflammatory myofibroblastic tumor, scirrhous carcinoma of the stomach, follicular dendritic cell sarcoma, inflammatory malignant fibrous histiocytoma, myofibroma/myofibromatosis, and solitary fibrous tumor of the stomach. CONCLUSIONS: Aggressive fibromatosis of the stomach is a rare spindle cell tumor that must be differentiated from a variety of conditions.

RGC32 induces epithelial-mesenchymal transition by activating the Smad/Sip1 signaling pathway in CRC.

Response gene to complement 32 (RGC32) is a transcription factor that regulates the expression of multiple genes involved in cell growth, viability and tissue-specific differentiation. However, the role of RGC32 in tumorigenesis and tumor progression in colorectal cancer (CRC) has not been fully elucidated. Here, we showed that the expression of RGC32 was significantly up-regulated in human CRC tissues versus adjacent normal tissues. RGC32 expression was significantly correlated with invasive and aggressive characteristics of tumor cells, as well as poor survival of CRC patients. We also demonstrated that RGC32 overexpression promoted proliferation, migration and tumorigenic growth of human CRC cells in vitro and in vivo. Functionally, RGC32 facilitated epithelial-mesenchymal transition (EMT) in CRC via the Smad/Sip1 signaling pathway, as shown by decreasing E-cadherin expression and increasing vimentin expression. In conclusion, our findings suggested that overexpression of RGC32 facilitates EMT of CRC cells by activating Smad/Sip1 signaling.

DcR3 induces epithelial-mesenchymal transition through activation of the TGF-ß3/SMAD signaling pathway in CRC.

Decoy receptor 3 (DcR3), a novel member of the tumor necrosis factor receptor (TNFR) family, was recently reported to be associated with tumorigenesis and metastasis. However, the role of DcR3 in human colorectal cancer (CRC) has not been fully elucidated. In this study, we found that DcR3 expression was significantly higher in human colorectal cancer tissues than in paired normal tissues, and that DcR3 expression was strongly correlated with tumor invasion, lymph node metastases and poor prognoses. Moreover, DcR3 overexpression significantly enhanced CRC cell proliferation and migration in vitro and tumorigenesis in vivo. Conversely, DcR3 knockdown significantly repressed CRC cell proliferation and migration in vitro, and DcR3 deficiency also attenuated CRC tumorigenesis and metastasis in vivo. Functionally, DcR3 was essential for TGF-ß3/SMAD-mediated epithelial-mesenchymal transition (EMT) of CRC cells. Importantly, cooperation between DcR3 and TGF-ß3/SMAD-EMT signaling-related protein expression was correlated with survival and survival time in CRC patients. In conclusion, our results demonstrate that DcR3 may be a prognostic biomarker for CRC and that this receptor facilitates CRC development and metastasis by participating in TGF-ß3/SMAD-mediated EMT of CRC cells.

AKAP-9 promotes colorectal cancer development by regulating Cdc42 interacting protein 4.

Our previous studies have shown that PRKA kinase anchor protein 9 (AKAP-9) is involved in colorectal cancer (CRC) cell proliferation and migration in vitro. However, whether or not AKAP-9 is important for CRC development or metastasis in vivo remains unknown. In the present study, we found that AKAP-9 expression was significantly higher in human colorectal cancer tissues than the paired normal tissues. In fact, AKAP-9 level correlated with the CRC infiltrating depth and metastasis. Moreover, the higher AKAP-9 expression was associated with the lower survival rate in patients. In cultured CRC cells, knockdown of AKAP-9 inhibited cell proliferation, invasion, and migration. AKAP-9 deficiency also attenuated CRC tumor growth and metastasis in vivo. Mechanistically, AKAP-9 interacted with cdc42 interacting protein 4 (CIP4) and regulated its expression. CIP4 levels were interrelated to the AKAP-9 level in CRC cells. Functionally, AKAP-9 was essential for TGF-ß1-induced epithelial-mesenchymal transition of CRC cells, and CIP4 played a critical role in mediating the function of AKAP-9. Importantly, CIP4 expression was significantly up-regulated in human CRC tissues. Taken together, our results demonstrated that AKAP-9 facilitates CRC development and metastasis via regulating CIP4-mediated epithelial-mesenchymal transition of CRC cells.

Long non-coding RNA MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation in colorectal cancer cells.

Our earlier findings indicate that the long non-coding RNA MALAT1 promotes colorectal cancer (CRC) cell proliferation, invasion and metastasis in vitro and in vivo by increasing expression of AKAP-9. In the present study, we investigated the molecular mechanism by which MALAT1 enhances AKAP9 expression in CRC SW480 cells. We found that MALAT1 interacts with both SRPK1 and SRSF1. MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation. Following MALAT1 knockdown, overexpression of SRPK1 was sufficient to restore SRSF1 phosphorylation and AKAP-9 expression to a level that promoted cell proliferation, invasion and migration in vitro. Conversely, SRPK1 knockdown after overexpression of MALAT1 in SW480 cells diminished SRSF1 phosphorylation and AKAP-9 expression and suppressed cell proliferation, invasion and migration in vitro. These findings suggest MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation in CRC cells. These results reveal a novel molecular mechanism by which MALAT1 regulates AKAP-9 expression in CRC cells.

NDRG1 attenuates epithelial-mesenchymal transition of nasopharyngeal cancer cells via blocking Smad2 signaling.

N-myc downstream-regulated gene 1 (NDRG1) has been implicated in tumorigenesis and metastasis in different cancers. However, its role in nasopharyngeal carcinoma remains unknown. We found that NDRG1 expression level was high in nasopharyngeal cancer 5-8F cells but low in 5-8F-LN cells with lymphatic metastasis potential. Knockdown of NDRG1 by shRNA promoted 5-8F cell proliferation, migration, and invasion in vitro and its tumorigenesis in vivo. Moreover, NDRG1 deficiency induced an epithelial-mesenchymal transition (EMT) of 5-8F cells as shown by an attenuation of E-cadherin and an induction of N-cadherin and vimentin expression. NDRG1 knockdown also enhanced Smad2 expression and phosphorylation. Smad2 signaling was attenuated in 5-8F cells but was significantly activated in 5-8F-LN cells. Knockdown of Smad2 restored E-cadherin but attenuated N-cadherin expression in NDRG1-deficient 5-8F cells, suggesting a reduction of EMT. Consistently, blockade of Smad2 in 5-8F-LN cells increased E-cadherin while diminishing N-cadherin and vimentin expression. These data indicate that Smad2 mediates the NDRG1 deficiency-induced EMT of 5-8F cells. In tumors derived from NDRG1-deficient 5-8F cells, E-cadherin expression was inhibited while vimentin and Smad2 were increased in a large number of cancer cells. Most importantly, NDRG1 expression was attenuated in human nasopharyngeal carcinoma tissues, resulted in a lower survival rate in patients. The NDRG1 was further decreased in the detached nasopharyngeal cancer cells, which was associated with a further reduced survival rate in patients with lymphatic metastasis. Taken together, these results demonstrated that NDRG1 prevents nasopharyngeal tumorigenesis and metastasis via inhibiting Smad2-mediated EMT of nasopharyngeal cells.

MALAT1 promotes colorectal cancer cell proliferation/migration/invasion via PRKA kinase anchor protein 9.

Our previous studies have shown that the 3' end of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is involved in colorectal cancer (CRC) cell proliferation and migration/invasion in vitro. The role and mechanism of MALAT1 in CRC metastasis in vivo, however, remain largely unknown. In the present study, we found that MALAT1 was up-regulated in human primary CRC tissues with lymph node metastasis. Overexpression of MALAT1 via RNA activation promoted CRC cell proliferation, invasion and migration in vitro, and stimulated tumor growth and metastasis in mice in vivo. Conversely, knockdown of MALAT1 inhibited CRC tumor growth and metastasis. MALAT1 regulated at least 243 genes in CRC cells in a genome-wide expression profiling. Among these genes, PRKA kinase anchor protein 9 (AKAP-9) was significantly up-regulated at both mRNA and protein levels. AKAP-9 was highly expressed in CRC cells with metastatic potential and human primary CRC tissues with lymph node metastasis, but not in normal cells or tissues. Importantly, knockdown of AKAP-9 blocked MALAT1-mediated CRC cell proliferation, migration and invasion. These data indicate that MALAT1 may promote CRC tumor development via its target protein AKAP-9.

The establishment of supramolecular immunobead real-time PCR and the identification of Cox-2 as a metastasis-related marker in colorectal carcinoma.

Cyclooxygenase-2 (Cox-2) is an inducible enzyme that converts arachidonic acid to prostaglandins, and it is hypothesized to induce carcinogenesis and metastasis in colorectal cancer. Our previous data also indicated that a higher expression level of Cox-2 was correlated with colorectal cancer metastasis. The Cox-2 protein was detected in the glandular cavity of colorectal cancer and the surrounding interstitial tissues. The usefulness of the Cox-2 gene as a gene therapy target and diagnostic marker remains unknown. In this study, a method using immuno-PCR and real-time PCR followed by supramolecular immunobead real-time PCR was established and used to detect the expression of Cox-2 in serum samples of nude mice with colorectal carcinoma. In addition, we established a Cox-2 gene stable knockdown colorectal cell line (SW480-EGFP-Cox-2 shRNA) using lentiviral vector-mediated RNA interference (RNAi) technology and established an imageable colorectal cancer metastasis mouse model. We found that the proliferation, invasion and tumorigenesis of SW480-EGFP-Cox-2 shRNA cells were attenuated compared with SW480 cells. In vivo experiments also demonstrated that angiogenesis in the Cox-2 knockdown colorectal cancer cells was decreased. The whole body optical imaging revealed that the SW480-EGFP-Cox-2 shRNA cells had an abrogated ability to develop metastases in the lymph nodes, lungs or liver in vivo. The improved immunobead PCR assay detected significantly lower Cox-2 protein levels in the serum samples of the SW480-EGFP-Cox-2 shRNA group compared with those of the SW480-EGFP-Cox-2-Ctrl shRNA group. In conclusion, our results indicated that the knockdown of Cox-2 expression suppressed the proliferation and invasion of colorectal cancer cells both in vitro and in vivo. This study also demonstrated that silencing Cox-2 in vivo reduced the metastastic potential of colorectal cancer. Thus, Cox-2 is a promising marker for the diagnosis of colorectal metastasis and a potential therapeutic target for colorectal cancer.

[Overexpression of response gene to complement-32 promotes cytoskeleton reorganization in SW480 cell line].

OBJECTIVE: To construct the recombinant plasmid pcDNA3.0-RGC32 and evaluate the effect of the response gene to complement-32 (RGC32) on cell cytoskeleton in vitro. METHODS: The full-length cDNA of RGC32 was obtained by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.0 to generate the recombinant plasmid pcDNA3.0-RGC32. After transfection of the recombinant plasmid into SW480 cells, the expression of RGC32 in the cells was detected by Western blotting. The cytoskeleton of SW480 cells was visualized before and after the transfection, and the changes in the cell migration ability was assessed by wound-healing assay. RESULTS: The recombinant plasmid pcDNA3.0-RGC32 was successfully constructed. The expression of RGC32 was significantly increased in SW480 cells after transfection with pcDNA3.0-RGC32. Before the transfection, the microfilaments of SW480 cells were few and short without obvious polarity, but after the transfection, the microfilaments were increased and elongated with also an obvious polarity, and the invasive structures of lamellae and lamellipodia occurred. The migration ability of the cells was enhanced after transfection with pcDNA3.0-RGC32. CONCLUSION: Overexpression of RGC32 can cause the reorganization of cytoskeleton and promotes the cell migration, which can be an important mechanism of RGC32 in promoting cancer metastasis.

Gene expression profile changes and possible molecular subtypes in differentiated-type nonkeratinizing nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a human malignant tumor with a high incidence and a poor prognosis in Southern China and South-eastern Asia. In this study, we comprehensively analyzed the gene expression profiles in 24 samples of primary differentiated-type nonkeratining NPC (DNK-NPC) tissues, 24 samples of normal nasopharyngeal tissues and 4 DNK-NPC cell lines using cDNA microarray technology and bioinformatics methods. We found expression level of some genes was wildly alerted in the DNK-NPC samples. In addition, our hierarchical clustering analysis revealed 2 distinctive subtypes of gene expression patterns in DNK-NPC tissue samples. The discriminator genes were identified using a signal-to-noise (S(2)N) algorithm by permuting of the data set 10,000 times. To further characterize the clinical relevance of the tumor subtypes, we evaluated a surrogate marker, CCND2, differentially expressed between the 2 tumor subgroups by using immunohistochemistry in an independent set of 137 DNK-NPC samples. CCND2 was highly expressed in the subgroups with "aggressive" features and was associated with T classification (p = 0.006) and clinical stage (p = 0.013). Patients with high level of CCND2 expression had poorer overall survival than those with low level (p = 0.034). Our results suggest that DNK-NPC can be classified into 2 subtypes based on gene expression patterns, which can be used in determining prognosis and treatment of the tumor.

[Toxicity of matrine in Kunming mice].

OBJECTIVE: To investigate the acute toxicity and assess the median lethal dose (LD50) of matrine in Kunming mice. METHODS: Matrine at different doses were administered in Kunming mice via intraperitoneal injection, and the toxic reactions and LD50 of matrine was observed and determined. RESULTS: The acute toxicity test of matrine indicated that the tolerable dose of matrine was above 80 mg/kg in Kunming mice, and the LD50 was 157.13 mg/kg (95%CI, 88.08-280.31 mg/kg). Morphological observation revealed degenerative changes of the nerve cells in the brain tissue of the mice. CONCLUSION: The nervous system is the main target organ by the toxicity of matrine.

Celecoxib enhances the inhibitory effect of cisplatin on Tca8113 cells in human tongue squamous cell carcinoma in vivo and in vitro.

BACKGROUND: Overexpression of cyclooxygenase-2 (COX-2) is associated with carcinogenesis, invasiveness, and metastasis of malignant tumors. Inhibition of COX-2 is one hot topic of research in prevention and treatment of malignant tumors. Because of the selective and specific inhibition on the activity of COX-2, the roles of celecoxib in prevention and treatment of tumors have attracted broad attention in recent years. In this study, we investigated the inhibitory effect of celecoxib combined with cisplatin on the proliferation of human tongue squamous cell carcinoma cell line Tca8113 in vivo and in vitro. METHODS: Human tongue squamous cell carcinoma tumor cells Tca8113 and a mouse model with Tca8113 cells were used to study the growth inhibition of cisplatin enhanced by celecoxib. Drug treatment of Tca8113 in vitro and mice bearing xenografts in vivo were used. The level of COX-2 expression was detected by Western blotting. Sensitivity of cells to drug treatment was analyzed by MTT assay. RESULTS: Treatment of Tca8113 cells with cisplatin (CDDP) had less effect on the expression of COX-2, whereas the COX-2 expression was significantly down-regulated after treatment with celecoxib alone or in combination with CDDP for 24 h. In addition, the combination of celecoxib with CDDP was also able to inhibit the Tca8113 line heterotransplanted in Balb/c nude mice. CONCLUSIONS: Those findings indicate that a low dose of celecoxib could augment CDDP-induced growth inhibition of Tca8113 cells and its xenograft in Balb/c nude mice.

[Celecoxib enhances the lethal effects of bleomycin in human tongue squamous carcinoma cell line Tca8113].

OBJECTIVE: To investigate the role of COX-2 inhibitor celecoxib in enhancing the lethal effects of bleomycin in Tca8113 cell line. METHODS: Tca8113 cells were treated with different concentrations of celecoxib and bleomycin for 24, 48, 72 h. Methyl thiazolyl tetrazolium assay was used to calculate cell growth inhibition rate and Jin Zheng Jun's method was used to evaluate the interaction of celecoxib and bleomycin on Tca8113 cells. Flow cytometry was used to evaluate the effects of combined use of celecoxib and bleomycin on cell cycle progress and apoptosis. RESULTS: Low dose of celecoxib (10 micromol/L, < IC(50)) combined with bleomycin showed synergism or additive lethal effect on Tca8113 cell line. Celecoxib could notably enhance the inhibitory effect of bleomycin on Tca8113 cells by blocking cell cycle progress and thus resulting in the increasing G(0)/G(1) cells [(60.93 +/- 0.32)%] distribution and inducing apoptosis [(1.87 +/- 0.11)%]. CONCLUSIONS: Low doses of celecoxib could significantly enhance the lethal effect of bleomycin on Tca8113 cells by inhibiting cell growth and proliferation through blocking cell cycle progress and inducing apoptosis. The ways of these interactions on inhibiting Tca8113 cell growth were synergistic or/and additive.

RGC-32 mediates transforming growth factor-beta-induced epithelial-mesenchymal transition in human renal proximal tubular cells.

Epithelial-mesenchymal transition (EMT) occurs in several disease states, including renal fibrosis and carcinogenesis. Myofibroblasts produced from EMT of renal tubular cells are responsible for the deposition of extracellular matrix components in a large portion of renal interstitial fibrosis. Transforming growth factor-beta (TGF-beta) plays an essential role in the EMT of renal tubular cells, but the molecular mechanism governing this process remains largely unknown. In this study, we found that RGC-32 (response gene to complement 32) is critical for TGF-beta-induced EMT of human renal proximal tubular cells (HPTCs). RGC-32 is not normally expressed in the HPTCs. However, TGF-beta stimulation markedly activates RGC-32 while inducing an EMT, as shown by the induction of smooth muscle alpha-actin (alpha-SMA) and extracellular matrix proteins collagen I and fibronectin, as well as the reduction of epithelial marker E-cadherin. TGF-beta function is mediated by several signaling pathways, but RGC-32 expression in HPTCs appears to be mainly regulated by Smad. Functionally, RGC-32 appears to mediate TGF-beta-induced EMT of HPTCs. Blockage of RGC-32 using short hairpin interfering RNA significantly inhibits TGF-beta induction of myofibroblast marker gene alpha-SMA while repressing the expression of E-cadherin. In contrast, overexpression of RGC-32 induces alpha-SMA expression while restoring E-cadherin. RGC-32 also inhibits the expression of another adherens junction protein, N-cadherin, suggesting that RGC-32 alone induces the phenotypic conversion of renal epithelial cells to myofibroblasts. Additional studies show that RGC-32 stimulates the production of extracellular matrix components fibronectin and collagen I. Mechanistically, RGC-32 induces EMT via the activation of other transcription factors such as Snail and Slug. RGC-32 knockdown inhibits the expression of Snail and Slug during TGF-beta-induced EMT. Taken together, our data demonstrate for the first time that RGC-32 plays a critical role in TGF-beta-induced EMT of renal tubular cells.

[Matrine-induced apoptosis in human colon adenocarcinoma SW620 cells].

OBJECTIVE: To investigate the effects of matrine on the cell cycle and apoptosis in human colon adenocarcinoma SW620 cells and explore the possible mechanisms. METHODS: The effect of matrine on cell proliferation was assessed using MTT assay, and the cell cycle arrest induced by matrine was determined by flow cytometry. The changes of cell morphology were observed through optical microscope, fluorescence microscope and electron microscope, and the cell apoptosis was detected using Annexin V-FITC apoptosis assay. RESULTS: Matrine inhibited the proliferation of SW620 cells in a dose- and time-dependent manner. Compared with the control group, the matrine-treated cells showed increased cell percentage arrested in G 0/G1 phase with decreased S-phase cells. Morphologically, the SW620 cells treated with matrine exhibited cell shrinkage, cell size reduction, plasma condensation, cytoplasmic vacuolar changes, and formation of apoptotic body with also the presence of the signet-ring cells, all typical of apoptotic cells. CONCLUSION: Matrine exposure of SW620 cells inhibits the cell proliferation, causes cell cycle arrest at G 0/G1 phase, and induces apoptosis in a dose- and time- dependent manner.