Klotho inhibits growth and promotes apoptosis in human lung cancer cell line A549

Background

Recently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was shown decreased expression in clear cell renal cell carcinoma (CCRCC), but the role of the down-expression of NDRG2 has not been described.

Methods

The NDRG2 recombinant adenovirus plasmid was constructed. The proliferation rate and NDRG2 expression of cell infected with recombinant plasmid were mesured by MTT, Flow cytometry analysis and western blot.

Results

The CCRCC cell A-498 re-expressed NDRG2 when infected by NDRG2 recombinant adenovirus and significantly decreased the proliferation rate. Fluorescence activated cell sorter analysis showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05). In addition, there were much more apoptotic cells in NDRG2-expressing cells than in the controls (P < 0.05). Moreover, upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E, whereas cyclinD2, cyclinD3 and cdk2 were not affected examined by western blot. Furthermore, we found that p53 could upregulate NDRG2 expression in A-498 cell.

Conclusions

We found that NDRG2 can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. p53 can up-regulate the expression of NDRG2. Our results showed that NDRG2 may function as a tumor suppressor in CCRCC.     

Tumour suppressor microRNA-584 directly targets oncogene Rock-1 and decreases invasion ability in human clear cell renal cell carcinoma

Background:

The purpose of this study was to identify new tumour suppressor microRNAs (miRs) in clear cell renal cell carcinoma (ccRCC), carry out functional analysis of their suppressive role and identify their specific target genes.

Methods:

To explore suppressor miRs in RCC, miR microarray and real-time PCR were performed using HK-2 and A-498 cells. Cell viability, invasion and wound healing assays were carried out for functional analysis after miR transfection. To determine target genes of miR, we used messenger RNA (mRNA) microarray and target scan algorithms to identify target oncogenes. A 3′UTR luciferase assay was also performed. Protein expression of target genes in ccRCC tissues was confirmed by immunohistochemistry and was compared with miR-584 expression in ccRCC tissues.

Results:

Expression of miR-584 in RCC (A-498 and 769-P) cells was downregulated compared with HK-2 cells. Transfection of miR-584 dramatically decreased cell motility. The ROCK-1 mRNA was inhibited by miR-584 and predicted to be target gene. The miR-584 decreased 3′UTR luciferase activity of ROCK-1 and ROCK-1 protein expression. Low expression of miR-584 in ccRCC tissues was correlated with high expression of ROCK-1 protein. The knockdown of ROCK-1 by siRNA inhibited cell motility.

Conclusion:

miR-584 is a new tumour suppressor miR in ccRCC and inhibits cell motility through downregulation of ROCK-1.     

Factor inhibiting HIF (FIH-1) promotes renal cancer cell survival by protecting cells from HIF-1α-mediated apoptosis

Background:

Clear cell renal cell carcinoma (CCRCC) is the commonest form of kidney cancer. Up to 91% have biallelic inactivation of VHL, resulting in stabilisation of HIF-α subunits. Factor inhibiting HIF-1 is an enzyme that hydroxylates HIF-α subunits and prevents recruitment of the co-activator CBP/P300. An important question is whether FIH-1 controls HIF activity in CCRCC.

Methods:

Human VHL defective CCRCC lines RCC10, RCC4 and 786–O were used to determine the role of FIH-1 in modulating HIF activity, using small interfering RNA knockdown, retroviral gene expression, quantitative RT–PCR, western blot analysis, Annexin V and propidium iodide labelling.

Results:

Although it was previously suggested that FIH-1 is suppressed in CCRCC, we found that FIH-1 mRNA and protein are actually present at similar levels in CCRCC and normal kidney. The FIH-1 inhibition or knockdown in the VHL defective CCRCC lines RCC10 and RCC4 (which express both HIF-1α and HIF-2α) resulted in increased expression of HIF target genes. In the 786-O CCRCC cell line, which expresses only HIF-2α, FIH-1 attenuation showed no significant effect on expression of these genes; introduction of HIF-1α resulted in sensitivity of HIF targets to FIH-1 knockdown. In RCC4 and RCC10, knockdown of FIH-1 increased apoptosis. Suppressing HIF-1α expression in RCC10 prevented FIH-1 knockdown from increasing apoptosis.

Conclusion:

Our results support a unifying model in which HIF-1α has a tumour suppressor action in CCRCC, held in check by FIH-1. Inhibiting FIH-1 in CCRCC could be used to bias the HIF response towards HIF-1α and decrease tumour cell viability.     

Serum 20S proteasome is elevated in patients with renal cell carcinoma and associated with poor prognosis

Background:

To date, no reliable serum marker for clear cell renal cell carcinoma (CCRCC) is available. The aim of this study was to evaluate the putative significance of circulating 20S proteasome levels.

Methods:

Preoperative 20S proteasome serum levels were determined in 113 CCRCC patients and 15 healthy controls by a sandwich enzyme-linked immunosorbent assay. Associations with CCRCC, pathological variables, disease-specific survival (DSS), and response to sunitinib were evaluated.

Results:

Median 20S proteasome levels were higher in CCRCC patients than in healthy controls (4.66 vs 1.52 μg ml⁻¹, P<0.0001). The area under the receiver operating characteristics curve curve was 87.1%. The 20S proteasome levels were associated with symptoms (P=0.0008), distant metastases (P=0.0011), grade (P=0.0247), and necrosis (P=0.0462). The 20S proteasome levels were identified as a prognostic factor for DSS in both univariable (hazards ratio 1.21, P<0.001) and multivariable (hazards ratio 1.17, P=0.0015) survival analysis. In patients responding to sunitinib, 20S proteasome levels were lower than in patients with stable disease and progressive disease.

Conclusion:

This study demonstrates for the first time that increased 20S proteasome levels are associated with CCRCC, advanced disease, and poor prognosis. Routine use of this marker may allow better diagnosis, risk stratification, risk-adjusted follow-up, and identification of patients with a greater likelihood of response to targeted therapy.     

Suppression of activin A signals inhibits growth of malignant pleural mesothelioma cells

Background:

Activins control the growth of several tumour types including thoracic malignancies. In the present study, we investigated their expression and function in malignant pleural mesothelioma (MPM).

Methods:

The expression of activins and activin receptors was analysed by quantitative PCR in a panel of MPM cell lines. Activin A expression was further analysed by immunohistochemistry in MPM tissue specimens (N=53). Subsequently, MPM cells were treated with activin A, activin receptor inhibitors or activin-targeting siRNA and the impact on cell viability, proliferation, migration and signalling was assessed.

Results:

Concomitant expression of activin subunits and receptors was found in all cell lines, and activin A was overexpressed in most cell lines compared with non-malignant mesothelial cells. Similarly, immunohistochemistry demonstrated intense staining of tumour cells for activin A in a subset of patients. Treatment with activin A induced SMAD2 phosphorylation and stimulated clonogenic growth of mesothelioma cells. In contrast, treatment with kinase inhibitors of activin receptors (SB-431542, A-8301) inhibited MPM cell viability, clonogenicity and migration. Silencing of activin A expression by siRNA oligonucleotides further confirmed these results and led to reduced cyclin D1/3 expression.

Conclusion:

Our study suggests that activin A contributes to the malignant phenotype of MPM cells via regulation of cyclin D and may represent a valuable candidate for therapeutic interference.     

Expression of CIP2A in renal cell carcinomas correlates with tumour invasion, metastasis and patients' survival

Background:

Cancerous inhibitor of protein phosphatase 2A (CIP2A) drives cellular transformation. The objective of this study was to detect the potential effects of CIP2A in renal cell carcinomas (RCCs).

Methods:

A total of 107 RCC patients were involved in the study. Cancerous inhibitor of protein phosphatase 2A expression was investigated by real-time PCR and immunohistochemistry. In vitro, we examined the expression of CIP2A and c-Myc and tested the migration and invasion capability of A498 and KRC/Y cells with scratch migration assay and Matrigel invasion assay after down-regulating CIP2A expression using siRNA.

Results:

Cancerous inhibitor of protein phosphatase 2A was over-expressed in RCC tissues. Clear cell RCC showed an even higher-CIP2A expression level than papillary or chromophobe RCC did. The CIP2A immunostaining level was positively correlated with primary tumour stage, lymph node metastasis, distant metastasis, TNM stage and histological grade (all P<0.05). High-CIP2A expression implied poor survival for patients (P<0.05). Cancerous inhibitor of protein phosphatase 2A depletion by siRNA down-regulated c-Myc expression and attenuated the migration and invasion of RCC cells.

Conclusion:

Higher-CIP2A expression positively correlates with the aggressive phenotype of RCCs, and predicts poor prognosis for patients. Cancerous inhibitor of protein phosphatase 2A may be a novel target for prevention and treatment of RCC metastasis and recurrence.     

Small nucleolar RNA 78 promotes the tumorigenesis in non-small cell lung cancer

Background

Accumulating evidence suggests that dysregulated snoRNA may play a role in the development of malignancy. In the present study, we investigated the role of SNORD78 in the tumorigenesis of non-small cell lung cancer (NSCLC).

Methods

We determined the expression level of SNORD78 in NSCLC tissues with quantitative real-time PCR and then studied its clinical significance. We explored the biological significance of SNORD78 with gain-and-loss-of-function analyses both in vitro and in vivo.

Results

A great upregulation of SNORD78 was observed in cancer tissues compared to their adjacent normal tissues. Meanwhile, patients with high SNORD78 expression have significantly poorer prognosis than those with low expression. Inhibition of SNORD78 suppressed the proliferation of NSCLC cells via inducing G0/G1 cell cycle arrest and apoptosis while SNORD78 overexpression promoted the cell proliferation. SNORD78 promoted invasion of NSCLC cells via inducing epithelial-mesenchymal-transition (EMT). SNORD78 was also obviously upregulated in cancer stem-like cells and is required for the self-renewal of NSCLC. The oncogenic activity of SNORD78 was also confirmed with in vivo data.

Conclusion

Our study identified that SNORD78 may be a potential therapeutic target for NSCLC.     

Absence of VHL gene alteration and high VEGF expression are associated with tumour aggressiveness and poor survival of renal-cell carcinoma

Background:

The von Hippel–Lindau gene (VHL) alteration, a common event in sporadic clear-cell renal-cell carcinoma (CCRCC), leads to highly vascularised tumours. Vascular endothelial growth factor (VEGF) is the major factor involved in angiogenesis, but the prognostic significance of both VHL inactivation and VEGF expression remain controversial. The aims of this study were to analyse the relationship between VHL genetic and epigenetic alterations, VHL expression and VEGF tumour or plasma expression, and to analyse their respective prognostic value in patients with CCRCC.

Methods:

A total of 102 patients with CCRCC were prospectively analysed. Alterations in VHL were determined by sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA. Expression of pVHL and VEGF was determined by immunohistochemistry. Plasma VEGF was measured by enzyme-linked immunosorbent assay (ELISA).

Results:

VHL mutation, deletion and promoter methylation were identified in 70, 76 and 14 cases, respectively. Overall, at least one VHL-gene alteration occurred in 91 cases (89.2%). Both VEGF tumour and plasma expression appeared to be decreased in case of VHL alteration. Median progression-free survival and CCRCC-specific survival were significantly reduced in patients with wild-type VHL or altered VHL and high VEGF expression, which, therefore, represent two markers of tumour aggressiveness in CCRCC.

Conclusion:

Stratifying CCRCCs according to VHL and VEGF status may help tailor therapeutic strategy.     

Contrasting effect of recombinant human erythropoietin on breast cancer cell response to cisplatin induced cytotoxicity

Background

Human recombinant erythropoietin (rHuEpo) that is used for the treatment of the chemotherapy-induced anaemia in cancer patients was shown to cause detrimental effects on the course of disease due to increased adverse events inflicting patient’s survival, potentially related to rHuEpo-induced cancer progression. In this study, we elucidate the effect of rHuEpo administration on breast cancer cell proliferation and gene expression after cisplatin (cDDP) induced cytotoxicity.

Materials and methods

Two breast carcinoma models, MCF-7 and MDA-MB-231 cell lines, were used differing in oestrogen (ER) and progesterone (PR) receptors and p53 status. Cells were cultured with or without rHuEpo for 24 h or 9 weeks and their growth characteristics after cDDP treatment were assessed together with expression of genes involved in the p53-signaling pathway.

Results

Short-term exposure of breast cancer cells to rHuEpo lowers their proliferation and reduces cDDP cytotoxic potency. In contrast, long-term exposure of MCF-7 cells to rHuEpo increases proliferation and predisposes MCF-7 cells to cDDP cytotoxicity, but has no effect on MDA-MB-231 cells. MDA-MB-231 cells show altered level of ERK phosphorylation, indicating involvement of MAPK signalling pathway. Gene expression analysis of p53-dependent genes and bcl-2 gene family members confirmed differences between long and short-term rHuEpo effects, indicating the most prominent changes in BCL2 and BAD expression.

Conclusions

Proliferation and survival characteristics of MCF-7 cells are reversely modulated by the length of the rHuEpo exposure. On the other hand, MDA-MB-231 cells are almost irresponsive to long-term rHuEpo, supposedly due to the mutated p53 and ER(+)/PR(−) status. The p53 and ER/PR status may predict tumour response on rHuEpo and cDDP treatment.     

Metadherin regulates proliferation and metastasis via actin cytoskeletal remodelling in non-small cell lung cancer

Background:

Metaderin (MTDH) protein is a novel component part of tight junction complex. The aim of this study was to investigate the correlation between MTDH and prognosis of patients and to explore the role of MTDH on NSCLC development and metastasis.

Methods:

Relative mRNA expression was evaluated by quantitative real-time PCR, and protein expression was detected using immunohistochemistry staining. The role of MTDH in cancer cell proliferation, migration and invasion was studied by modulation of MTDH expression in NSCLC cell lines. These functions of MTDH were further confirmed in vivo.

Results:

In NSCLC, low MTDH protein expression was correlated with lymph node metastasis, TNM stage and decreased OS (P=0.001, 0.011 and 0.013, respectively). Overexpression of MTDH reduced anchorage-independent and -dependent growth through arresting cell cycle, inhibited migration and invasion in vitro and further suppressed tumorigenesis, tumour growth and metastasis in vivo. Knockdown of MTDH expression increased cell invasiveness. MTDH overexpression reversed pro-metastatic actin cytoskeleton remodelling and inhibited EMT, supporting that MTDH has a key role on cancer proliferation and metastasis.

Conclusions:

MTDH has an important role in NSCLC proliferation and metastasis and provides potential in predicting metastasis and prognosis for patients with NSCLC.     

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma

Background:

Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3β positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-κB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3β and to assess the anti-cancer effect of GSK-3β inhibition in RCC.

Methods:

Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3β in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT–PCR, BrDU incorporation and MTS assays to study the effect of GSK-3β inactivation on renal cancer cell proliferation and survival.

Results:

We detected aberrant nuclear accumulation of GSK-3β in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-κB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells.

Conclusions:

Our results show nuclear accumulation of GSK-3β as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.     

miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC)

Background:

Hypopharyngeal squamous cell carcinoma (HSCC) is an aggressive malignancy with one of the worst prognoses among all head and neck cancers. Greater understanding of the pertinent molecular oncogenic pathways could help improve diagnosis, therapy, and prevention of this disease. The aim of this study was to identify tumour-suppressive microRNAs (miRNAs), based on miRNA expression signatures from clinical HSCC specimens, and to predict their biological target genes.

Methods:

Expression levels of 365 human mature miRNAs from 10 HSCC clinical samples were screened using stem-loop real-time quantitative PCR. Downregulated miRNAs were used in cell proliferation assays to identify a tumour-suppressive miRNA. Genome-wide gene expression analyses were then performed to identify the target genes of the tumour-suppressive miRNA.

Results:

Expression analysis identified 11 upregulated and 31 downregulated miRNAs. Gain-of-function analysis of the downregulated miRNAs revealed that miR-489 inhibited cell growth in all head and neck cancer cell lines examined. The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene. Knockdown of PTPN11 resulted in the inhibition of cell proliferation in head and neck SCC cells.

Conclusion:

Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.     

Apoptosis of bladder transitional cell carcinoma T24 cells induced by adenovirus-mediated inducible nitric oxide synthase gene transfection

Objectives：To investigate the effects of adenovirus-mediated inducible nitric oxide synthase gene transfection on bladder transitional cell carcinoma T24 cells,and to provide novel insights and approaches to clinical therapies against bladder transitional cell carcinoma.Methods：Firstly,construct recombinant adenovirus vector pAd-iNOS of iNOS,followed by transfection of pAd-iNOS into HECK293 packaging cells.Thirdly,harvest recombinant adenovirus rAd-iNOS after amplification and purification procedures.Finally,transfect the recombinant adenovirus rAd-iNOS into human bladder carcinoma T24 cells and examine the effect of rAd-iNOS transfection on apoptosis of T24 and possible mechanism.Results：As shown by this study,the recombinant adenovirus rAd-iNOS was constructed successfully.The virus titer was 5.8×108 PFU/mL and recombinant was verified by PCR analysis.Transfection of adenovirus rAd-iNOS into T24 cells could induce secretion of high NO concentration,P53 protein expression upregulation,as well as promotion ofT24 cell apoptosis.Conclusions：The transfection of human bladder carcinoma T24 cells from recombinant adenovirus rAdiNOS was confirmed to induce intracellular iNOS over-expression,high production of NO,up-regulation of intracellular P53 expression and promotion of cell apoptosis.     

Lack of correlation of stem cell markers in breast cancer stem cells

Background:

Various markers are used to identify the unique sub-population of breast cancer cells with stem cell properties. Whether these markers are expressed in all breast cancers, identify the same population of cells, or equate to therapeutic response is controversial.

Methods:

We investigated the expression of multiple cancer stem cell markers in human breast cancer samples and cell lines in vitro and in vivo, comparing across and within samples and relating expression with growth and therapeutic response to doxorubicin, docetaxol and radiotherapy.

Results:

CD24, CD44, ALDH and SOX2 expression, the ability to form mammospheres and side-population cells are variably present in human cancers and cell lines. Each marker identifies a unique rather than common population of cancer cells. In vivo, cells expressing these markers are not specifically localized to the presumptive stem cell niche at the tumour/stroma interface. Repeated therapy does not consistently enrich cells expressing these markers, although ER-negative cells accumulate.

Conclusions:

Commonly employed methods identify different cancer cell sub-populations with no consistent therapeutic implications, rather than a single population of cells. The relationships of breast cancer stem cells to clinical parameters will require identification of specific markers or panels for the individual cancer.