Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress

Chinese hamster ovary (CHO) cells are regarded as one of the most commonly used mammalian hosts, which decreases the productivity due to loss in culture viability. Overexpressing antiapoptosis genes in CHO cells was developed as a means of limiting cell death upon exposure to environmental insults. Glucose‐regulated protein 78 (GRP78) is traditionally regarded as a major ER chaperone that participates in protein folding and other cell processes. It is also a potent antiapoptotic protein and plays a critical role in cell survival, proliferation, and metastasis. In this study, the impact of GRP78 on CHO cells in response to environmental insults such as serum deprivation and oxidative stress was investigated. First, it was confirmed that CHO cells were very sensitive to environmental insults. Then, GRP78 overexpressing CHO cell line was established and exposed to serum deprivation and H₂O₂. Results showed that GRP78 engineering increased the viability and decreased the apoptosis of CHO cells. The survival advantage due to GRP78 engineering could be mediated by suppression of caspase‐3 involved in cell death pathways in stressed cells. Besides, GRP78 engineering also enhanced yields of antibody against transferrin receptor in CHO cells. GRP78 should be a potential application in the biopharmaceutical industries.     

Skp2 is over-expressed in breast cancer and promotes breast cancer cell proliferation

The F box protein Skp2 is oncogenic. Skp2 and Skp2B, an isoform of Skp2 are overexpressed in breast cancer. However, little is known regarding the mechanism by which Skp2B promotes the occurrence and development of breast cancer. Here, we determined the expression and clinical outcomes of Skp2 in breast cancer samples and cell lines using breast cancer database, and investigated the role of Skp2 and Skp2B in breast cancer cell growth, apoptosis and cell cycle arrest. We obtained Skp2 is significantly overexpressed in breast cancer samples and cell lines, and high Skp2 expression positively correlated with poor prognosis of breast cancer. Both Skp2 and Skp2B could promote breast cancer cell proliferation, inhibit cell apoptosis, change the cell cycle distribution and induce the increased S phase cells and therefore induce cell proliferation in breast cancer cells. Moreover, the 2 isoforms could both suppress PIG3 expression via independent pathways in the breast cancer cells. Skp2 suppressed p53 and inhibited PIG3-induced apoptosis, while Skp2B attenuated the function of PIG3 by inhibiting PHB. Our results indicate that Skp2 and Skp2B induce breast cancer cell development and progression, making Skp2 and Skp2B potential molecular targets for breast cancer therapy.     

M2 macrophages contribute to cell proliferation and migration of breast cancer

Breast cancer is a kind of malignant tumor that severely threatens women's lives and health worldwide. Tumor-associated macrophages (TAMs) have been reported to mediate tumor progression, while the mechanism still needs further identification. In this study, we found that M2 macrophages promoted increased cell proliferation and migration as well as reduced expression of interferon regulatory factor 7 (IRF7) and increased the expression of miR-1587 in breast cancer cells. Overexpression of IRF7 or miR-1587 knockdown reversed M2 macrophage-induced cell proliferation and migration as well as tumor growth in vivo. Mechanistically, miR-1587 targeted the 3ʹ-untranslated region (3ʹ-UTR) of IRF7 mRNA to regulate its protein expression leading to tumor progression. Collectively, this study revealed that the miR-1587/IRF7 axis mediates M2 macrophage-induced breast cancer progression, and this sheds light on further clinical therapy for breast cancer by targeting TAMs as well as the miR-1587/IRF7 axis.     

Overexpression of Hsc70 promotes proliferation,migration, and invasion of human glioma cells

Migration and invasion are often recognized as the main reasons for the high recurrence and death rates of glioma and limit the efficacy of surgery and other antitumor therapies. In this study, we found over activation of heat shock cognate protein 70 (Hsc70) in human glioma specimens, which was closely related to glioma grade. We investigated whether Hsc70 induced the migration and invasion of glioma cells. Wound healing and transwell migration assay were used to determine the migration and invasion ability of human glioma U251 and U87 cells, in which the expression of Hsc70 was knocked down by small interfering RNA. Western blot analysis was performed to determine the expression of FAK-Src signaling in malignant glioma cells. The results showed that Hsc70 deficiency significantly retarded migration and invasion and reduced the phosphorylation of FAK, Src, and Pyk2 in U251 and U87 cells. Overall, our results indicate that the migration and invasion capacity of human brain glioma cells is at least partly induced by Hsc70-dependent activation of FAK-Src signaling.     

Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line

Attrora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma （ESCC）. It has been demonstrated that cells overexpressing Attrora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Attrora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly （ADPribose） polymerase （PARP） in Attrora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A＇s effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.     

Local anesthetics counteract cell proliferation and migration of human triple-negative breast cancer and melanoma cells

In different retrospective studies, a protective role of regional anesthetics in reducing cancer recurrence after surgery was indicated. Accordingly, it has been previously demonstrated a protective effect of anesthetics in breast cancer cells and in other types of cancer. On the other hand, how anesthetics influence cancer needs in-depth investigations. For this purpose, two different human cancer cell lines, MDA-MB-231, triple-negative breast cancer, and A375, melanoma, were used in this study. By means of Western blotting and immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick end labeling analyses, the signal transduction pathways activated by the anesthetics, such as ropivacaine and levobupivacaine, were analyzed. The data obtained demonstrated that both anesthetics are able to counteract cell proliferation by positively modulating cell death signaling and by decreasing cell proliferation and survival pathways.     

Aquaporin-5: a marker protein for proliferation and migration of human breast cancer cells

Aquaporin (AQP) is a family of transmembrane proteins for water transport. Recent studies revealed that AQPs are likely to play a role in tumor progression and invasion. We aimed to examine the potential role of AQP5 in the progression of human breast cancer cells. Expression of AQP5 mRNA and protein was seen in human breast cancer cell line (both MCF7 and MDA-MB-231) by RT-PCR and immunoblotting analysis. Immunoperoxidase labeling of AQP5 was observed at ductal epithelial cells of human breast tissues. In benign tumor, AQP5 labeling was mainly seen at the apical domains of ductal epithelial cells. In contrast, in invasive ductal carcinoma, prominent AQP5 labeling was associated with cancer cells, whereas some ducts were unlabeled and apical polarity of AQP5 in ducts was lost. Cell proliferation (BrdU incorporation assay) and migration of MCF7 cells were significantly attenuated by lentivirus-mediated AQP5-shRNA transduction. Hyperosmotic stress induced by sorbitol treatment (100 mM, 24 h) reduced AQP5 expression in MCF7 cells, which was also associated with a significant reduction in cell proliferation and migration. Taken together, prominent AQP5 expression in breast cancer cells with the loss of polarity of ductal epithelial cells was seen during the progression of breast carcinoma. shRNA- or hyperosmotic stress-induced reduction in AQP5 expression of MCF7 cells was associated with significantly reduced cell proliferation and migration. In conclusion, AQP5 overexpression is likely to play a role in cell growth and metastasis of human breast cancer and could be a novel target for anti-breast cancer treatment.     

Overexpression of VCC-1 gene in human hepatocellular carcinoma cells promotes cell proliferation and invasion

Vascular endothelial growth factor-correlated chemokine 1 （VCC-1）, a novel chemokine, is hypothesized to be associated with carcinogenesis. VCC-1 is expressed in hepatocellular carcinoma （HCC） cells, but its func- tion remains unknown. To investigate the molecular effects of VCC-1 on HCC cells, the HCC cell line SMMC7721 was stably transfected with the recombinant plasmid pcDNA3.1/VCC-1. Our data demonstrated that overexpression of VCC-1 in SMMC7721 cells significantly enhanced the cellular proliferation, invasive ability, and tumor growth, when compared with both empty vector control cells and parental cells. These results strongly suggest that VCC-1 plays an important role in SMMC7721 invasion and tumor growth, and indicate that VCC-1 may serve as a potential biomarker for anti-HCC therapies.     

Constitutively active erythropoietin receptor expression in breast cancer cells promotes cellular proliferation and migration through a MAP-kinase dependent pathway

The role of erythropoietin receptor (EpoR) expression in tumor cells and the potential of EpoR-mediated signaling to contribute to cellular proliferation and invasiveness require further characterization. To determine whether EpoR expression and activation in tumor cells modulates intracellular signal transduction to promote cellular proliferation and migration, we employed a novel experimental model using human breast cancer cells engineered to stably express a constitutively active EpoR-R129C variant. EpoR-R129C expression resulted in increased cellular proliferation and migration of breast cancer cells and these effects were associated with significantly increased Epo-induced phosphorylation of ERK1/2, AKT and c-Jun-NH2-kinase (SAPK/JNK) proteins. Expression of the constitutively active EpoR-R129C receptor promoted the proliferation and migration of breast cancer cells via activation of ERK- and SAPK/JNK-dependent signaling pathways, respectively. These findings suggest that EpoR over-expression and activation in breast cancer cells has the potential to contribute to tumor progression by promoting the proliferation and invasiveness of the neoplastic cells.     

Upregulation of HYAL1 expression in breast cancer promoted tumor cell proliferation, migration, invasion and angiogenesis

Hyaluronic acid (HA) is a component of the Extra-cellular matrix (ECM), it is closely correlated with tumor cell growth, proliferation, metastasis and angiogenesis, etc. Hyaluronidase (HAase) is a HA-degrading endoglycosidase, levels of HAase are elevated in many cancers. Hyaluronidase-1 (HYAL1) is the major tumor-derived HAase. We previously demonstrated that HYAL1 were overexpression in human breast cancer. Breast cancer cells with higher HAase expression, exhibited significantly higher invasion ability through matrigel than those cells with lower HAase expression, and knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis. Here, to further elucidate the function of HYAL1 in breast cancer, we investigated the consequences of forcing HYAL1 expression in breast cancer cells by transfection of expression plasmid. Compared with control, HYAL1 up-regulated cells showed increased the HAase activity, and reduced the expression of HA in vitro. Meantime, upregulation of HYAL1 promoted the cell growth, migration, invasion and angiogenesis in vitro. Moreover, in nude mice model, forcing HYAL1 expression induced breast cancer cell xenograft tumor growth and angiogenesis. Interestingly, the HA expression was upregulated by forcing HYAL1 expression in vivo. These findings suggested that HYAL1-HA system is correlated with the malignant behavior of breast cancer.     

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.     

Overexpression of human osteopontin increases cell proliferation and migration in human embryo kidney-293 cells

Malignant tumors are characterized by dysregulated cell growth and the metastasis of secondary tumors. Numerous studies have documented that osteopontin (OPN) plays a key role in regulating tumor progression and metastasis. Here, we show that the overexpression of OPN in human embryo kidney-293 cells significantly increases both the level of cell proliferation, by provoking the G₁/S transition, and the level of cell migration in vitro. These findings suggest that augmented OPN contributes to cell growth and motility. Inhibiting OPN or the pathway it stimulates may therefore represent a novel approach for the treatment of primary tumors and associated metastases.     

Mitochondrial manganese-superoxide dismutase expression in ovarian cancer: role in cell proliferation and response to oxidative stress

Superoxide dismutases (SODs) are important antioxidant enzymes responsible for the elimination of superoxide radical (O(2)(-)). The manganese-containing SOD (Mn-SOD) has been suggested to have tumor suppressor function and is located in the mitochondria where the majority of O(2)(-) is generated during respiration. Although increased reactive oxygen species (ROS) in cancer cells has long been recognized, the expression of Mn-SOD in cancer and its role in cancer development remain elusive. The present study used a human tissue microarray to analyze Mn-SOD expression in primary ovarian cancer tissues, benign ovarian lesions, and normal ovary epithelium. Significantly higher levels of Mn-SOD protein expression were detected in the malignant tissues compared with normal tissues (p < 0.05). In experimental systems, suppression of Mn-SOD expression by small interfering RNA caused a 70% increase of superoxide in ovarian cancer cells, leading to stimulation of cell proliferation in vitro and more aggressive tumor growth in vivo. Furthermore, stimulation of mitochondrial O(2)(-) production induced an increase of Mn-SOD expression. Our findings suggest that the increase in Mn-SOD expression in ovarian cancer is a cellular response to intrinsic ROS stress and that scavenging of superoxide by SOD may alleviate the ROS stress and thus reduce the simulating effect of ROS on cell growth.     

Spondin 2 promotes the proliferation,migration and invasion of gastric cancer cells

Spondin 2 (SPON2), a member of the Mindin F‐Spondin family, identifies pathogens, activates congenital immunity and promotes the growth and adhesion of neurons as well as binding to their receptors, but its role in promoting or inhibiting tumour metastasis is controversial. Here, we investigated its expression levels and mechanism of action in gastric cancer (GC). Western blotting and GC tissue arrays were used to determine the expression levels of SPON2. ELISAs were performed to measure the serum levels of SPON2 in patients with GC. Two GC cell lines expressing low levels of SPON2 were used to analyse the effects of regulating SPON2 expression on proliferation, migration, invasion, the cell cycle and apoptosis. The results revealed that SPON2 was highly expressed in GC tissues from patients with relapse or metastasis. The levels of SPON2 in sera of patients with GC were significantly higher compared with those of healthy individuals and patients with atrophic gastritis. Knockdown of SPON2 expression significantly inhibited the proliferation, migration and invasion of GC cells in vitro and in vivo. Down‐regulation of SPON2 arrested the cell cycle in G1/S, accelerated apoptosis through the mitochondrial pathway and inhibited the epithelial‐mesenchymal transition by blocking activation of the ERK1/2 pathway. In summary, this study suggests that SPON2 acts as an oncogene in the development of GC and may serve as a marker for the diagnosing GC as well as a new therapeutic target for GC.     

Arachidonic acid promotes FAK activation and migration in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n-6 polyunsaturated fatty acid that is present in an esterified form in cell membrane phospholipids, and it might be present in the extracellular microenvironment. In particular, AA promotes MAPK activation and mediates the adhesion of MDA-MB-435 breast cancer cells to type IV collagen. However, the signal transduction pathways mediated by AA have not been studied in detail. Our results demonstrate that stimulation of MDA-MB-231 breast cancer cells with AA promotes an increase in the phoshorylation of Src and FAK, as revealed by site-specific antibodies that recognized the phosphorylation state of Src at Tyr-418, and of FAK at tyrosine-397 and in vitro kinase assays. In addition, AA also induces an increase in the migration of MDA-MB-231 cells. In contrast, AA does not induce phosphorylation of FAK and an increase in cell migration of non-tumorigenic epithelial cells MCF10A. Inhibition of Gi/Go proteins, LOX and Src activity prevent FAK activation and cell migration. In conclusion, our results demonstrate, for the first time, that Gi/Go proteins, LOX and Src play an important role in FAK activation and cell migration induced by AA in MDA-MB-231 breast cancer cells.