AZD2014 Radiosensitizes Oral Squamous Cell Carcinoma by Inhibiting AKT/mTOR Axis and Inducing G1/G2/M Cell Cycle Arrest

Background

Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms in Taiwan. Activation of the mTOR signaling pathway has been linked to decreased radiation responsiveness in human oral cancer, thus it limits efficacy of radiotherapy. To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.

Methods

We isolated primary tumor cells from OSCC tissues and cell lines. AZD2014 was administered with and without ionizing radiation. The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses. Western blotting was used to detect protein expression.

Results

Combination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity. Pre-treatment with AZD2014 in irradiated oral cancer cells induced tumor cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing caspase-3 and LC3 in primary OSCC cells.

Conclusions

These findings suggest that in irradiated OSCC cells, co-treatment with AZD2014, which targets mTORC1 and mTORC2 blockade, is an effective radiosensitizing strategy for oral squamous cell carcinoma.     

Galectin-3 and Beclin1/Atg6 genes in human cancers: using cDNA tissue panel, qRT-PCR, and logistic regression model to identify cancer cell biomarkers

Background

Cancer biomarkers are sought to support cancer diagnosis, predict cancer patient response to treatment and survival. Identifying reliable biomarkers for predicting cancer treatment response needs understanding of all aspects of cancer cell death and survival. Galectin-3 and Beclin1 are involved in two coordinated pathways of programmed cell death, apoptosis and autophagy and are linked to necroptosis/necrosis. The aim of the study was to quantify galectin-3 and Beclin1 mRNA in human cancer tissue cDNA panels and determine their utility as biomarkers of cancer cell survival.

Methods and Results

A panel of 96 cDNAs from eight (8) different normal and cancer tissue types were used for quantitative real-time polymerase chain reaction (qRT-PCR) using ABI7900HT. Miner2.0, a web-based 4- and 3- parameter logistic regression software was used to derive individual well polymerase chain reaction efficiencies (E) and cycle threshold (Ct) values. Miner software derived formula was used to calculate mRNA levels and then fold changes. The ratios of cancer to normal tissue levels of galectin-3 and Beclin1 were calculated (using the mean for each tissue type). Relative mRNA expressions for galectin-3 were higher than for Beclin1 in all tissue (normal and cancer) types. In cancer tissues, breast, kidney, thyroid and prostate had the highest galectin-3 mRNA levels compared to normal tissues. High levels of Beclin1 mRNA levels were in liver and prostate cancers when compared to normal tissues. Breast, kidney and thyroid cancers had high galectin-3 levels and low Beclin1 levels.

Conclusion

Galectin-3 expression patterns in normal and cancer tissues support its reported roles in human cancer. Beclin1 expression pattern supports its roles in cancer cell survival and in treatment response. qRT-PCR analysis method used may enable high throughput studies to generate molecular biomarker sets for diagnosis and predicting cancer treatment response.     

Tetracycline Inhibits Local Inflammation Induced by Cerebral Ischemia via Modulating Autophagy

Background

Tetracycline exerts neuroprotection via suppressing the local inflammation induced by cerebral ischemia. However, the underlying mechanism is not completely clear.

Methodology/Principal Findings

The mRNA and protein expressions of tumor necrosis factor α and interleukin 6 and the number of activated microglia were measured to detect the inflammatory process in the ischemic hemisphere. The key proteins of nuclear factor kappa B pathway and the binding activity of nuclear factor kappa B were also measured. Two key components of autophagy, Beclin 1 and LC3, were detected by western blotting. Pretreatment with tetracycline inhibited the mRNA and protein expressions of tumor necrosis factor α and interleukin 6 and decreased the numbers of activated and phagocytotic microglia. Tetracycline down regulated the total and phosphorylated expressions of IKK, IκB and p65 (P<0.05). The autophagy inhibitor, 3-methyladenine, inhibited inflammation and activation of nuclear factor kappa B pathway. The levels of Beclin 1 and LC3 were decreased by 3-methyladenine and tetracycline.

Conclusions/Significance

Our data suggested that pretreatment of tetracycline may inhibit autophagy in the ischemic stroke brain and then suppress the inflammatory process via inhibiting the activation of nuclear factor kappa B pathway.     

Transforming Growth Factor TGFβ Increases Levels of Microtubule-Associated Protein MAP1S and Autophagy Flux in Pancreatic Ductal Adenocarcinomas

Background and Aim

Autophagy is a cellular process to regulate the turnover of misfolded/aggregated proteins or dysfunctional organelles such as damaged mitochondria. Microtubule-associated protein MAP1S (originally named C19ORF5) is a widely-distributed homologue of neuronal-specific MAP1A and MAP1B with which autophagy marker light chain 3 (LC3) was originally co-purified. MAP1S bridges autophagic components with microtubules and mitochondria through LC3 and positively regulates autophagy flux from autophagosomal biogenesis to degradation. The MAP1S-mediated autophagy suppresses tumorigenesis as suggested in a mouse liver cancer model and in prostate cancer patients. The TGFβ signaling pathway plays a central role in pancreatic tumorigenesis, and high levels of TGFβ suggest a tumor suppressive function and predict a better survival for some patients with resectable pancreatic ductal adenocarcinoma. In this study, we try to understand the relationship between TGFβ and MAP1S-mediated autophagy in pancreatic ductal adenocarcinoma.

Methods

We collected the tumor and its adjacent normal tissues from 33 randomly selected patients of pancreatic ductal adenocarcinomas to test the association between TGFβ and autophagy markers MAP1S and LC3. Then we tested the cause and effect relation between TGFβ and autophagy markers in cultured pancreatic cancer cell lines.

Results

Here we show that levels of TGFβ and autophagy markers MAP1S and LC3 are dramatically elevated in tumor tissues from patients with pancreatic ductal adenocarcinomas. TGFβ increases levels of MAP1S protein and enhances autophagy flux.

Conclusion

TGFβ may suppress the development of pancreatic ductal adenocarcinomas by enhancing MAP1S-mediated autophagy.     

Protective Effect of Sevoflurane Postconditioning against Cardiac Ischemia/Reperfusion Injury via Ameliorating Mitochondrial Impairment,Oxidative Stress and Rescuing Autophagic Clearance

Background and Purpose

Myocardial infarction leads to heart failure. Autophagy is excessively activated in myocardial ischemia/reperfusion (I/R) in rats. The aim of this study is to investigate whether the protection of sevoflurane postconditioning (SPC) in myocardial I/R is through restored impaired autophagic flux.

Methods

Except for the sham control (SHAM) group, each rat underwent 30 min occlusion of the left anterior descending coronary (LAD) followed by 2 h reperfusion. Cardiac infarction was determined by 2,3,5-triphenyltetrazolium chloride triazole (TTC) staining. Cardiac function was examined by hemodynamics and echocardiography. The activation of autophagy was evaluated by autophagosome accumulation, LC3 conversion and p62 degradation. Potential molecular mechanisms were investigated by immunoblotting, real-time PCR and immunofluorescence staining.

Results

SPC improved the hemodynamic parameters, cardiac dysfunction, histopathological and ultrastructural damages, and decreased myocardial infarction size after myocardial I/R injury (P < 0.05 vs. I/R group). Compared with the cases in I/R group, myocardial ATP and NAD⁺ content, mitochondrial function related genes and proteins, and the expressions of SOD2 and HO-1 were increased, while the expressions of ROS and Vimentin were decreased in the SPC group (P < 0.05 vs. I/R group). SPC significantly activated Akt/mTOR signaling, and inhibited the formation of Vps34/Beclin1 complex via increasing expression of Bcl2 protein (P < 0.05 vs. I/R group). SPC suppressed elevated expressions of LC3 II/I ratio, Beclin1, Atg5 and Atg7 in I/R rat, which indicated that SPC inhibited over-activation of autophagy, and promoted autophagosome clearance. Meanwhile, SPC significantly suppressed the decline of Opa1 and increases of Drp1 and Parkin induced by I/R injury (P < 0.05 vs. I/R group). Moreover, SPC maintained the contents of ATP by reducing impaired mitochondria.

Conclusion

SPC protects rat hearts against I/R injury via ameliorating mitochondrial impairment, oxidative stress and rescuing autophagic clearance.     

14-3-3τ Regulates Beclin 1 and Is Required for Autophagy

Background

Beclin 1 plays an essential role in autophagy; however, the regulation of Beclin 1 expression remains largely unexplored. An earlier ChIP-on-chip study suggested Beclin 1 could be an E2F target. Previously, we also reported that 14-3-3τ regulates E2F1 stability, and is required for the expression of several E2F1 target genes. 14-3-3 proteins mediate many cellular signaling processes, but its role in autophagy has not been investigated. We hypothesize that 14-3-3τ could regulate Beclin 1 expression through E2F1 and thus regulate autophagy.

Methods and Findings

Using the RNAi technique we demonstrate a novel role for one of 14-3-3 isoforms, 14-3-3τ, in the regulation of Beclin 1 expression and autophagy. Depletion of 14-3-3τ inhibits the expression of Beclin 1 in many different cell lines; whereas, upregulation of 14-3-3τ induces Beclin 1. The regulation is physiologically relevant as an extracellular matrix protein tenascin-C, a known 14-3-3τ inducer, can induce Beclin 1 through 14-3-3τ. Moreover, rapamycin-induced, serum free-induced and amino acid starvation-induced autophagy depends on 14-3-3τ. We also show the expression of Beclin 1 depends on E2F, and E2F can transactivate the Beclin 1 promoter in a promoter reporter assay. Upregulation of Beclin 1 by 14-3-3τ requires E2F1. Depletion of E2F1, like 14-3-3τ, also inhibits autophagy.

Conclusion

Taken together, this study uncovers a role for 14-3-3τ in Beclin 1 and autophagy regulation probably through regulation of E2F1.     

Important Role of Autophagy in Endothelial Cell Response to Ionizing Radiation

Objectives

Vasculature damage is an important contributor to the side-effects of radiotherapy. The aim of this study is to provide insights into the radiobiology of the autophagic response of endothelial cells.

Methods and Materials

Human umbilical vascular endothelial cells (HUVEC) were exposed to 2 Gy of ionizing radiation (IR) and studied using confocal microscopy and western blot analysis, at 4 and 8 days post-irradiation. The role of autophagy flux in HUVEC radio-sensitivity was also examined.

Results

IR-induced accumulation of LC3A+, LC3B+ and p62 cytoplasmic vacuoles, while in double immunostaining with lysosomal markers (LAMP2a and CathepsinD) repression of the autophagolysosomal flux was evident. Autophagy-related proteins (ATF4, HIF1α., HIF2α, Beclin1) were, however, induced excluding an eventual repressive effect of radiation on autophagy initiating protein expression. Exposure of HUVEC to SMER28, an mTOR-independent inducer of autophagy, enhanced proLC3 and LC3A, B-I protein expression and accelerated the autophagic flux. Pre-treatment of HUVEC with SMER28 protected against the blockage of autophagic flux induced by IR and conferred radio-resistance. Suppression of LC3A/LC3B proteins with siRNAs resulted in radio-sensitization.

Conclusions

The current data provide a rationale for the development of novel radioprotection policies targeting the autophagic pathway.     

Interleukin-22 Alleviated Palmitate-Induced Endoplasmic Reticulum Stress in INS-1 Cells through Activation of Autophagy

Objective

Stimulation with saturated fatty acids has been shown to induce oxidative stress and endoplasmic reticulum (ER) stress in β cells and has been recognized as an important component of the pathogenesis of type 2 diabetes (T2D). Interleukin-22 (IL-22) plays a critical role in preventing β cells from oxidative and ER stress, and autophagy is associated with the survival and function of β cells. However, whether IL-22 alleviates cellular stress through activation of autophagy is unclear. In this study, we investigated the effects of IL-22 on rat insulin-secreting cells and the mechanisms underlying IL-22 and lipotoxicity-induced oxidative and ER stress in vitro.

Methods

The levels of reactive oxygen species (ROS) were detected by flow cytometry and fluorescence microscopy. The protein expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), microtubule-associated protein light chain 3B (LC3B) and Bcl-2-interacting myosin-like coiled-coil protein (Beclin-1) were evaluated by western blot. Transmission electron microscopy was utilized to observe the process of autophagy.

Results

Palmitate induced increased levels of ROS and the overexpression of GRP78 and CHOP, whereas these effects were partly reversed by treatment with IL-22. Furthermore, IL-22 upregulated the protein expression of Beclin-1 and the conversion of LC3B-I to LC3B-II. Moreover, the aforementioned effects were partly suppressed by treating cells with 3-methyladenine (3-MA), an autophagy inhibitor.

Conclusions

Our results suggest that IL-22 alleviated the oxidative and ER stress induced by palmitate, which was likely mediated by autophagy. These findings could facilitate the development of novel therapeutic strategies to suppress the progression of T2D.     

Inhibition of Autophagy Contributes to Ischemic Postconditioning-Induced Neuroprotection against Focal Cerebral Ischemia in Rats

Background

Ischemic postconditioning (IPOC), or relief of ischemia in a stuttered manner, has emerged as an innovative treatment strategy to reduce programmed cell death, attenuate ischemic injuries, and improve neurological outcomes. However, the mechanisms involved have not been completely elucidated. Recent studies indicate that autophagy is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. This study aims to determine the role of autophagy in IPOC-induced neuroprotection against focal cerebral ischemia in rats.

Methodology/Principal Findings

A focal cerebral ischemic model with permanent middle cerebral artery (MCA) occlusion plus transient common carotid artery (CCA) occlusion was established. The autophagosomes and the expressions of LC3/Beclin 1/p62 were evaluated for their contribution to the activation of autophagy. We found that autophagy was markedly induced with the upregulation of LC3/Beclin 1 and downregulation of p62 in the penumbra at various time intervals following ischemia. IPOC, performed at the onset of reperfusion, reduced infarct size, mitigated brain edema, inhibited the induction of LC3/Beclin 1 and reversed the reduction of p62 simultaneously. Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effects induced by IPOC. Conversely, autophagy inhibitor 3-methyladenine (3-MA) attenuated the ischemic insults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.

Conclusions/Significance

The present study suggests that inhibition of the autophagic pathway plays a key role in IPOC-induced neuroprotection against focal cerebral ischemia. Thus, pharmacological inhibition of autophagy may provide a novel therapeutic strategy for the treatment of stroke.     

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis

Purpose

Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.

Methods

Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5–5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.

Results

Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced.

Discussion

Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.     

Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly

Background

Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient’s prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules ‘(i.e. nucleotides, aminoacids, fatty acids). In cancer, Malic enzyme (ME) and ATP-citrate lyase (ACLY) are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC).

Material and Methods

ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient’s survival.

Results

Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite.

Conclusion

In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.     

Inhibition of Breast Cancer Cell Proliferation and In Vitro Tumorigenesis by a New Red Apple Cultivar

Purpose

The aim of this study was to evaluate the antiproliferative activity in breast cancer cells and the inhibition of tumorigenesis in pre-neoplastic cells of a new apple cultivar with reddish pulp, called the Pelingo apple.

Methods

The antiproliferative activity was evaluated in MCF-7 and MDA-MB-231 human breast cancer cells. The inhibition of tumorigenesis was performed in JB6 promotion-sensitive (P+) cells.

Results

Results showed that Pelingo apple juice is characterized by a very high polyphenol content and strongly inhibited breast cancer cell proliferation. Its antiproliferative activity was found to be higher than the other five apple juices tested. Pelingo juice induced cell accumulation in the G2/M phase of the cell cycle and autophagy through overexpression of p21, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) activity and an increase in lipidated microtubule-associated protein-1 light chain-3 beta (LC3B). Remarkably, Pelingo juice inhibited the 12-o-tetra-decanoyl-phorbol-13-acetate (TPA)-induced tumorigenesis of JB6 P+ cells, suppressing colony formation in semi-solid medium and TPA-induced ERK1/2 phosphorylation.

Conclusions

Our data indicate that the Pelingo apple is rich in food components that can markedly inhibit in vitro tumorigenesis and growth of human breast cancer cells and could provide natural bioactive non-nutrient compounds, with potential chemopreventive activity.     

Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

Objectives

The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients.

Materials and Methods

Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence differences were recognized as somatic mutations.

Results

Somatic mutations in the D-loop of mtDNA were identified in 75 (62.5%) oral squamous cell carcinoma patients and most of them occurred in the poly-C tract. Although there were no significant differences in demographic and tumor-related features between participants with and without somatic mutation, the mutation group had a better survival rate (5 year disease-specific survival rate: 64.0% vs. 43.0%, P = 0.0266).

Conclusion

Somatic mutation in D-loop of mtDNA was associated with a better survival in oral squamous cell carcinoma patients.     

PD-L1 and Survival in Solid Tumors: A Meta-Analysis

Background

Numerous agents targeting PD-L1/PD-1 check-point are in clinical development. However, the correlation between PD-L1expression and prognosis of solid tumor is still in controversial. Here, we elicit a systematic review and meta-analysis to investigate the potential value of PD-L1 in the prognostic prediction in human solid tumors.

Methods

Electronic databases were searched for studies evaluating the expression of PD-L1 and overall survival (OS) of patients with solid tumors. Odds ratios (ORs) from individual studies were calculated and pooled by using a random-effect model, and heterogeneity and publication bias analyses were also performed.

Results

A total of 3107 patients with solid tumor from 28 published studies were included in the meta-analysis. The median percentage of solid tumors with PD-L1 overexpression was 52.5%. PD-L1 overexpression was associated with worse OS at both 3 years (OR = 2.43, 95% confidence interval (CI) = 1.60 to 3.70, P < 0.0001) and 5 years (OR = 2.23, 95% CI = 1.40 to 3.55, P = 0.0008) of solid tumors. Among the tumor types, PD-L1 was associated with worse 3 year-OS of esophageal cancer, gastric cancer, hepatocellular carcinoma, and urothelial cancer, and 5 year-OS of esophageal cancer, gastric cancer and colorectal cancer.

Conclusions

These results suggest that expression of PD-L1 is associated with worse survival in solid tumors. However, the correlations between PD-L1 and prognosis are variant among different tumor types. More studies are needed to investigate the clinical value of PD-L1 expression in prognostic prediction and treatment option.     

Prognostic Value of Basic Fibroblast Growth Factor (bFGF) in Lung Cancer: A Systematic Review with Meta-Analysis

Background

Basic fibroblast growth factor (bFGF) is known to stimulate angiogenesis and thus to influence the proliferation, migration and survival of tumor cells. Many studies examined the relationship between human bFGF overexpression and survival in lung cancer patients, but the results have been mixed. To systematically summarize the clinical prognostic function of bFGF in lung cancer, we performed this systematic review with meta-analysis.

Method

Studies were identified by an electronic search of PubMed, EMBASE, China National Knowledge Infrastructure and Wanfang databases, including publications prior toAugust 2014. Pooled hazard ratios (HR) for overall survival (OS) were aggregated and quantitatively analyzed by meta-analysis.

Results

Twenty-two studies (n = 2154) were evaluated in the meta-analysis. Combined HR suggested that bFGF overexpression had an adverse impact on survival of patients with lung cancer(HR = 1.202,95%CI, 1.022–1.382). Our subgroup analysis revealed that the combined HR evaluating bFGF expression on OS in operable non-small cell lung cancer (NSCLC) was 1.553 (95%CI, 1.120–1.986); the combined HR in small cell lung cancer (SCLC) was 1.667 (95%CI, 1.035–2.299). There was no significant impact of bFGF expression on survival in advanced NSCLC.

Conclusion

This meta-analysis showed that bFGF overexpression is a potential indicator of worse prognosis for patients with operable NSCLC and SCLC, but is not associated with outcome in advanced NSCLC. The data suggests that high bFGF expression is highly related to poor prognosis. Nevertheless,more high-quality studies should be performed in order to provide additional evidence for the prognostic value of bFGF in lung cancer.     

Expression of Sirtuin 1 and 2 Is Associated with Poor Prognosis in Non-Small Cell Lung Cancer Patients

Background

Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology.

Material and Methods

Protein levels of SIRT1 and SIRT2 were determined in non-small cell lung cancer (NSCLC) cell lines and primary tumors from 105 patients. Changes in proliferation were assessed after SIRT1 and SIRT2 downregulation in lung cancer cell lines using siRNA-mediated technology or tenovin-1, a SIRT1 and SIRT2 inhibitor.

Results

High SIRT1 and SIRT2 protein levels were found in NSCLC cell lines compared with non-tumor lung epithelial cells. The expression of SIRT1 and SIRT2 proteins was also significantly higher in lung primary tumors than in normal tissue (P<0.001 for both sirtuins). Stronger nuclear SIRT1 staining was observed in adenocarcinomas than in squamous cell carcinomas (P=0.033). Interestingly, in NSCLC patients, high SIRT1 and SIRT2 expression levels were associated with shorter recurrence-free survival (P=0.04 and P=0.007, respectively). Moreover, the combination of high SIRT1 and SIRT2 expression was an independent prognostic factor for shorter recurrence-free survival (P=0.002) and overall survival (P=0.022). In vitro studies showed that SIRT1 and/or SIRT2 downregulation significantly decreased proliferation of NSCLC.

Conclusions

Our results support the hypothesis that SIRT1 and SIRT2 have a protumorigenic role in lung cancer, promoting cell proliferation. Moreover, the expression of these proteins is associated with poor prognosis in NSCLC patients and may help to identify those NSCLC patients with high risk of recurrence that could benefit from adjuvant therapy after resection.     

Tenascin-C,a Prognostic Determinant of Esophageal Squamous Cell Carcinoma

Background

Tenascin-C, an adhesion modulatory extracellular matrix molecule, is highly expressed in numerous human malignancies; thus, it may contribute to carcinogenesis and tumor progression. We explored the clinicopathological significance of Tenascin-C as a prognostic determinant of esophageal squamous cell carcinoma (ESCC).

Methods

In ESCC patient tissues and cell lines, the presence of isoforms were examined using western blotting. We then investigated Tenascin-C immunohistochemical expression in 136 ESCC tissue samples. The clinical relevance of Tenascin-C expression and the correlation between Tenascin-C expression and expression of other factors related to cancer-associated fibroblasts (CAFs) were also determined.

Results

Both 250 and 350 kDa sized isoforms of Tenascin-C were expressed only in esophageal cancer tissue not in normal tissue. Furthermore, both isoforms were also identified in all of four CAFs derived from esophageal cancer tissues. Tenascin-C expression was remarkably higher in ESCC than in adjacent non-tumor esophageal epithelium (p < 0.001). Tenascin-C expression in ESCC stromal fibroblasts was associated with patient’s age, tumor (pT) stage, lymph node metastasis, clinical stage, and cancer recurrence. Tenascin-C expression in cancer cells was correlated with an increase in tumor-associated macrophage (TAM) population, cancer recurrence, and hypoxia inducible factor1α (HIF1α) expression. Moreover, Tenascin-C overexpression in cancer cells and stromal fibroblasts was an independent poor prognostic factor for overall survival (OS) and disease-free survival (DFS). In the Cox proportional hazard regression model, Tenascin-C overexpression in cancer cells and stromal fibroblasts was a significant independent hazard factor for OS and DFS in ESCC patients in both univariate and multivariate analyses. Furthermore, Tenascin-C expression in stromal fibroblasts of the ESCC patients was positively correlated with platelet-derived growth factor α (PDGFRα), PDGFRβ, and smooth muscle actin (SMA) expression. The 5-year OS and DFS rates were remarkably lower in patients with positive expressions of both Tenascin-C and PDGFRα (p < 0.001), Tenascin-C and PDGFRβ (p < 0.001), Tenascin-C and SMA (p < 0.001), Tenascin-C and fibroblast activation protein (FAP) (p < 0.001), and Tenascin-C and fibroblast-stimulating protein-1 (FSP1) (p < 0.001) in ESCC stromal fibroblasts than in patients with negative expressions of both Tenascin-C and one of the abovementioned CAF markers.

Conclusion

Our results show that Tenascin-C is a reliable and significant prognostic factor in ESCC. Tenascin-C may thus be a potent ESCC therapeutic target.     

Rational incorporation of selenium into temozolomide elicits superior antitumor activity associated with both apoptotic and autophagic cell death

Background

The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure.

Principal Findings

This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se.

Conclusions

Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug.     

Reversine suppresses oral squamous cell carcinoma via cell cycle arrest and concomitantly apoptosis and autophagy

Background

The effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated.

Methods

Effects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence.

Results

The results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy.

Conclusions

Taken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.