Activation of TRPV4 Induces Exocytosis and Ferroptosis in Human Melanoma Cells.

TRPV4 (transient receptor potential vanilloid 4), a calcium permeable TRP ion channel, is known to play a key role in endocytosis. However, whether it contributes to exocytosis remains unclear. Here, we report that activation of TRPV4 induced massive exocytosis in both melanoma A375 cell and heterologous expression systems. We show here that, upon application of TRPV4-specific agonists, prominent vesicle priming from endoplasmic reticulum (ER) was observed, followed by morphological changes of mitochondrial crista may lead to cell ferroptosis. We further identified interactions between TRPV4 and folding/vesicle trafficking proteins, which were triggered by calcium entry through activated TRPV4. This interplay, in turn, enhanced TRPV4-mediated activation of folding and vesicle trafficking proteins to promote exocytosis. Our study revealed a signaling mechanism underlying stimulus-triggered exocytosis in melanoma and highlighted the role of cellular sensor TRPV4 ion channel in mediating ferroptosis.

Taurine attenuates acrylamide-induced axonal and myelinated damage through the Akt/GSK3ß-dependent pathway.

Acrylamide (ACR), formed during the Maillard reaction induced by high temperature in food processing, is one of the main causes of neurodegenerative diseases. Taurine, a free intracellular ß-amino acid, is characterized by many functions, including antioxidation, anti-inflammatory, and neuroprotective properties. This promotes its application in the treatment of neurodegenerative diseases. In this study, the neuroprotective effects of taurine against ACR-induced neurotoxicity and the potential underlying mechanisms were explored. Rats were intoxicated with ACR and injected with taurine in different groups for totally 2 weeks between January and July 2017. Electron microscopic analysis was used to observe the changes in tissues of the rats. Meanwhile, the levels of proteins including p-Akt, p-GSK3ß, SIM312, and MBP were detected by Western blot. Furthermore, the GSK3ß phosphorylation in taurine-treated dorsal root ganglion (DRG) with ACR was examined in the presence of the Akt inhibitor, MK-2206. The analysis of behavioral performances and electron micrographs indicated that taurine treatment significantly attenuated the toxic manifestations induced by ACR and stimulated the growth of axons and the medullary sheath, which was associated with the activation of the Akt/GSK3ß signaling pathway. Mechanistically, it was found that taurine activated GSK3ß, leading to significant recovery of the damage in ACR-induced sciatic nerves. Furthermore, MK-2206, an inhibitor of Akt, was applied in DRG cells, suggesting that taurine-induced GSK3ß phosphorylation was Akt dependent. Our findings demonstrated that taurine attenuated ACR-induced neuropathy in vivo, in an Akt/GSK3ß-dependent manner. This confirmed the treatment with taurine to be a novel strategy against ACR-induced neurotoxicity.

Prognostic analysis of Chinese patients with metastasis renal cell cancer receiving sorafenib: results from a multicenter long-term follow-up retrospective study.

The effects of sorafenib for Chinese patients with metastatic renal cell cancer (mRCC) were evaluated to figure out the relationship between clinical variables and prognosis. The data were analyzed retrospectively from six comprehensive cancer centers in Northeast China. All cases were diagnosed as mRCC histopathologically without exception. Patients were taken 400 mg sorafenib orally twice daily until progression of disease or intolerable toxic reaction occurred. Overall survival (OS), progression-free survival (PFS), and the influence of clinical variables on survival were appointed as main outcome measures. Clinical data were analyzed using SPSS statistical software. P<0.05 was considered as statistically significant. A total of 131 patients were available for survival analysis. The median follow-up periods were 16.9 months, and the median OS and PFS were 16.1 months and 10.5 months, respectively. Univariate analysis showed that Eastern Cooperative Oncology Group performance status (ECOG PS), metastatic sites, and previous therapy were significantly associated with OS, whereas PFS was merely associated with ECOG PS and previous therapy. The multivariate analysis suggested that ECOG PS, metastatic sites, and previous therapy were the independent prognostic factors for OS, and ECOG PS and previous therapy as the independent prognostic factors for PFS. In the subgroup analysis for patients with visceral metastasis, the prognosis of patients with lung metastasis alone was better than those cases with liver metastasis alone or multiple organs metastasis. In our study, sorafenib shows a higher curative activity for patients with mRCC in Northeast China. ECOG PS, metastatic lesions, and previous therapy may be important parameters for OS and PFS prediction. Lung metastases alone may be a more sensitive indicator for sorafenib than other organ metastases.

Small breast epithelial mucin tumor tissue expression is associated with increased risk of recurrence and death in triple-negative breast cancer patients.

BACKGROUND: Small breast epithelial mucin (SBEM) has been implicated in tumor genesis and micrometastasis in breast cancer. Triple-negative breast cancer (TNBC) was characterized by high incidence in young women,early relapse and a very poor prognosis. The aim of this study was to evaluate the association of SBEM expression in tissues of TNBC with disease-free survival (DFS) and overall survival (OS). METHODS: SBEM protein expression was detected in 87 available formalin-fixed paraffin-embedded (FFPE) tissue specimens from TNBC patients by means of immunohistochemistry (IHC). We analyzed the correlation between the SBEM protein expression and DFS and OS during a 5 year follow-up period, respectively. And a SBEM cut-off value of prognosis was established associated with DFS and OS. SBEM was analyzed against other risk factors in multivariate analysis. RESULTS: SBEM 3+ score was cut-off value of prognosis and significantly correlated with DFS (p = 0.000) and OS (p = 0.001) in TNBC patients. There was a marked associations (p <0.05) between SBEM 3+ score and tumor size, grade, node status, TNM stage and Ki67. Multivariate analysis showed that patients with SBEM 3+ represented a higher risk of recurrence and mortality than those with a lower SBEM expression (HR = 3.370 with p = 0.008 for DFS and HR = 4.185 with p = 0.004 for OS). CONCLUSIONS: SBEM is an independent risk predictor and may offer utility as a prognostic marker in TNBC patients.

[Clinical controlled trial of first-line treatment for advanced kidney cancer].

OBJECTIVE: To estimate the efficacies of different first-line treatments for advanced stage kidney cancer. METHODS: For this observation controlled trial, a total of 82 cases with advanced stage kidney cancer from 2006 to 2011 were recruited. They were divided into 3 groups and accepted gemcitabine plus interleukin-2 (IL-2) (Group A), oxaliplatin plus capecitabine (Group B) or sorafenib alone (Group C). RESULTS: Among them, 76 patients had complete data. The overall response rates of A-C groups were 39.3% (11/28), 37.0% (10/27) and 38.1% (8/21) respectively. And there was no significant difference (χ(2) = 0.029, P = 0.986). And their progression-free survival (PFS) rates were 9.1 (95%CI: 7.9 - 10.3), 7.5(95%CI: 5.5 - 9.5) and 10.9 (95%CI: 10.5 - 11.3) months respectively. And there were significant differences (P = 0.013). Average daily treatment costs were 490, 498 and 501 Chinese yuan respectively. And there was no significant difference (P = 1.240). Because of toxicity, 2 and 3 cases withdrew in Groups A and B respectively. CONCLUSION: Gemcitabine plus IL-2 and oxaliplatin plus capecitabine have similar early efficacies and tolerance profiles for the patients who can not accept sorafenib as first-line treatment.

Small breast epithelial mucin (SBEM) has the potential to be a marker for predicting hematogenous micrometastasis and response to neoadjuvant chemotherapy in breast cancer.

To investigate the potential role of small breast epithelial mucin (SBEM) as a marker for detecting hematogenous micrometastasis in breast cancer and explore its clinical significance in neoadjuvant chemotherapy. SBEM protein expression in 82 tissue specimens of primary breast cancer was detected using immunohistochemistry (IHC), and SBEM expression in peripheral blood (PB) samples of 109 primary breast cancer patients (94 cases at stage I-III, 15 cases at stage IV) was detected by flow cytometry (FCM) and reverse transcription polymerase chain reaction (RT-PCR). Moreover, SBEM mRNA expression was monitored by quantification real-time PCR (QPCR) before and after 3 cycles' neoadjuvant chemotherapy. SBEM expression correlated with tumor node metastasis (TNM) staging and lymph node metastasis at both mRNA and protein levels. SBEM expression in PB of breast cancer patients was markedly higher than that of healthy donors and other cancer patients. SBEM was found expressed in PB of 50 cases among 94 cases at stage I-III and expressed in PB of 11 cases among 15 cases at stage IV. After 3 cycles' neoadjuvant chemotherapy, SBEM expression levels were significantly down-regulated in up to 58% breast cancer patients. SBEM has the potential to be a specific marker for predicting hematogenous micrometastasis and response to neoadjuvant chemotherapy in breast cancer.

Study on relationship between angiogenesis and micrometastases of peripheral blood in breast cancer.

PURPOSE: To investigate the relationship between microvessel density, expression of vascular endothelial growth factor A (VEGF-A) and micrometastases in peripheral blood of patients with breast cancer. METHOD: Microvessel density (MVD) and expression of VEGF-A were detected by immunohistochemistry S-P. Nested RT-PCR was introduced to detect the expression of hMAM mRNA in peripheral blood of all cases. RESULT: Average MVD was 28.95 +/- 6.95 microvessels/100x and positive rate of VEGF-A was 64.0% (32/50) in 50 cases with breast cancer. MVD count and expression of VEGF-A were related to tumor size, metastasis of axillary lymph nodes and clinical stages (P < 0.05), independent of age and histological classification (P > 0.05). The positive rate of hMAM mRNA in peripheral blood was 34.0% (17/50), which correlated with lymphatic metastasis and clinical stages (P < 0.05), independent of pathological category, menopause and hormone receptor (P > 0.05). MVD count and positive rate of VEGF-A in breast cancer with positive expression of hMAM mRNA was obviously higher than those without hMAM mRNA expression (chi (2) = 5.766, P = 0.032; t = 5.37, P = 0.002). CONCLUSIONS: MVD count and positive expression of VEGF-A closely correlated to hMAM mRNA released from tumor cells in the circulation. hMAM mRNA is expected to become a valuable marker for further study on micrometastases of breast cancer.

[Relation of lymphatic microvessel density detected by monoclonal antibody D2-40 with VEGF-C expression in breast cancer].

OBJECTIVE: To investigate the relationship of lymphatic micovessel density (LMVD) detected by monoclonal antibody D2-40) with the VEGF-C expression in human breast cancer. METHODS: Tissue samples of 102 breast cancers, 25 breast fibroadenomas and 10 normal breasts were collected. Immunohistochemical staining was used to detected the lymphatic micovessels with monoclonal antibody D2-40. The expression of VEGF-C was detect by SP immunohistochemistry, and VEGF-C mRNA by hybridization in situ. RESULTS: In 102 breast cancers, the positive rate of D2-40 was 76.5% (78/102), higher than that in the breast fibroadenomas. LMVD in the periphery of breast cancer was 30.1 lymphatic microvessels per x 100 field of vision, which was significantly higher than that in the central area of the tumors (P = 0.000). The LMVD in the periphery of the breast cancers was correlated with the number of metastatic lymph nodes (r = 0.964, P < 0.01). The positive rates of VEGF-C protein and mRNA were 55.9% (57/102) and 59.8% (61/102), respectively, significantly higher than that in the breast fiberoadenomas and normal breast tissues (chi2 = 11.653, P = 0.003; chi2 = 10.345, P = 0.006), and were significantly correlated with the status of lymph node metastasis, clinical stage and the expressions of c-erbB-2 and p53 protein (P < 0.05). Both of VEGF-C protein and mRNA were significantly correlated with LMVD detected by D2-40 (P < 0.05), especially with the LMVD in the periphery of breast cancers (P < 0.05). CONCLUSION: The monoclonal antibody D240 can be used to detect the lymphatic endothelium in human breast cancer. The lymphatic microvessel density in the periphery of breast cancer is correlated with the lymph node metastasis and expression of VEGF-C. Therefore, VEGF-C may play a significant role in the lymphangiogenesis leading to metastasis of breast cancer.

CD147 regulates vascular endothelial growth factor-A expression, tumorigenicity, and chemosensitivity to curcumin in hepatocellular carcinoma.

CD147, also named as extracelluar matrix metalloproteinase inducer (EMMPRIN), has been proved to be involved in several aspects of tumor progression. In addition to its ability to induce vascular endothelial growth factor (VEGF) production, it confers resistance to some chemotherapeutic drugs. To investigate the possible role of CD147 in the mouse hepatocarcinoma cell line Hepa1-6 with no metastatic potential in the lymph nodes, we used RNA interference (RNAi) approach to silence CD147 expression. The results showed that silencing of CD147 in Hepa1-6 cells significantly impeded the expression of VEGF-A at both mRNA and protein levels. The siRNA-treated cells exhibited significantly decreased growth ability when compared with control cells. Colony formation of CD147 deficient cells was dramatically inhibited in soft agar, and tumorigenicity was reduced in nude mice. Furthermore, the downregulation of CD147 expression also sensitized cells to be more sensitive to curcumin. These results suggested that CD147 might be a potential target for therapeutic antitumor drugs.

Genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells.

The recent finding that acrylamide (AA), a carcinogen in animal experiments and a probable human carcinogen, is formed in foods during cooking raises human health concerns. The relevance of dietary exposure for humans is still under debate. The purpose of the study was to evaluate the possible genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells, a cell line of great relevance to detect genotoxic/antigenotoxic substances, using single cell gel electrophoresis (SCGE) assay and micronucleus test (MNT). In order to clarify the underlying mechanism(s) we evaluated the intracellular generation of reactive oxygen species (ROS) and the level of oxidative DNA damage by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The involvement of glutathione (GSH) in the AA-induced oxidative stress was examined through treatment with buthionine sulfoximine (BSO) to deplete GSH. The results indicate that AA caused DNA strand breaks and increase in frequency of MN in HepG2 cells in a dose-dependent manner. The possible mechanism underlies the increased levels of ROS, depletion of GSH and increase of 8-OHdG formation in HepG2 cells treated with AA. We conclude that AA exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.