The principal Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1) is strongly associated with nasopharyngeal carcinoma (NPC), a prevalent cancer in China. The epidermal growth factor receptor (EGFR) is important in carcinogenesis, as it is a ubiquitously expressed receptor tyrosine kinase. Signal transducer and activator of transcription 3 (STAT3) is a master transcriptional regulator in proliferation and apoptosis. Our previous study demonstrated that the nuclear EGFR could bind to the cyclin D1 promoter directly in the presence of LMP1, and the correlation between EGFR and STAT3 in NPC remains to be further explored. Here, we have shown that the interaction of EGFR and STAT3 increased in the nucleus in the presence of LMP1. LMP1 promoted both EGFR and STAT3 binding to the promoter region of cyclin D1, in turn, enhancing the promoter activity of cyclin D1. Furthermore, we demonstrated that both transcriptional activity and mRNA levels of cyclin D1 were decreased by small molecule interference of EGFR and STAT3 activity. These findings may provide a novel linkage between the EGFR and STAT3 signaling pathways and the activation of cyclin D1 by LMP1 in the carcinogenesis of NPC.
EGFR; STAT3; Cyclin D1; Epstein–Barr virus; Latent membrane protein 1; Nasopharyngeal carcinoma
The effectiveness of different breast cancer follow-up procedures to decrease breast cancer mortality are still an object of debate, even if intensive follow-up by imaging modalities is not recommended by international guidelines since 1997. We conducted a systematic review of surveillance procedures utilized, in the last ten years, in phase III randomized trials (RCTs) of adjuvant treatments in early stage breast cancer with disease free survival as primary endpoint of the study, in order to verify if a similar variance exists in the scientific world. Follow-up modalities were reported in 66 RCTs, and among them, minimal and intensive approaches were equally represented, each being followed by 33 (50%) trials. The minimal surveillance regimen is preferred by international and North American RCTs (P = 0.001) and by trials involving more than one country (P = 0.004), with no relationship with the number of participating centers (P = 0.173), with pharmaceutical industry sponsorship (P = 0.80) and with trials enrolling > 1000 patients (P = 0.14). At multivariate regression analysis, only geographic location of the trial was predictive for a distinct follow-up methodology (P = 0.008): Western European (P = 0.004) and East Asian studies (P = 0.010) use intensive follow-up procedures with a significantly higher frequency than international RCTs, while no differences have been detected between North American and international RCTs. Stratifying the studies according to the date of beginning of patients enrollment, before or after 1998, in more recent RCTs the minimal approach is more frequently followed by international and North American RCTs (P = 0.01), by trials involving more than one country (P = 0.01) and with more than 50 participating centers (P = 0.02). It would be highly desirable that in the near future breast cancer follow-up procedures will be homogeneous in RCTs and everyday clinical settings.
Breast cancer; Follow-up; Phase III clinical trial; Systematic review
To investigate the potential dosimetric and clinical benefits of Deep Inspiration Breath-Hold (DIBH) technique during radiotherapy of breast cancer compared with Free Breathing (FB).
Eight left-sided breast cancer patients underwent a supervised breath hold during treatment. For each patient, two CT scans were acquired with and without breath hold, and virtual simulation was performed for conventional tangential fields, utilizing 6 or 15 MV photon fields. The resulting dose–volume histograms were calculated, and the volumes of heart/lung irradiated to given doses were assessed. The left anterior descending coronary artery (LAD) mean and maximum doses were calculated, together with tumour control probability (TCP) and normal tissue complication probabilities (NTCP) for lung and heart.
For all patients a reduction of at least 16% in lung mean dose and at least 20% in irradiated pulmonary volumes was observed when DIBH was applied. Heart and LAD maximum doses were decreased by more than 78% with DIBH. The NTCP values for pneumonitis and long term cardiac mortality were also reduced by about 11% with DIBH. The NTCP values for pericarditis were zero for both DIBH and FB.
Delivering radiation in DIBH conditions the dose to the surrounding normal structures could be reduced, in particular heart, LAD and lung, due to increased distance between target and heart, and to reduced lung density.
Deep inspiration breath-hold gating radiotherapy; Breast cancer radiotherapy; NTCP; TCP
According to the International Multidisciplinary Classification of Lung Adenocarcinoma (LAD) by International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) in 2011, the diagnosis of LAD is changing from simple morphology into a comprehensive multidisciplinary classification. The aim of this study is to detect the expression of Notch-1 and analyze its clinicopathological or prognostic significance in different histological subtypes of Lung Adenocarcinomas (LADs).
Western blot and Semi-quantitative Reverse transcription-polymerase chain reaction (RT-PCR) assays, as well as immunohisitochemistry, were performed to detect the expression of Notch-1 in LAD cells and tissue samples. Kaplan-Meier and multivariate Cox regression analyses were performed to evaluate the correlation of Notch-1 expression with clinicopathological factors and prognosis of LAD patients.
The expression level of Notch-1 protein in LAD cell lines or tissues was significantly lower than that in normal human bronchial epithelial cell line (16HBE) or nontumor tissues (P < 0.05). By statistical analyses, it was observed that negative Notch-1 expression was significantly associated with advanced clinical stage (P = 0.001) and lymph node metastasis (P = 0.026) in LAD patients. Also, the recurrence rate of Notch-1-positive group was higher than the Notch-1-negative group (P = 0.001), and patients with positive Notch-1 expression have a prolonged progression of overall survival (P = 0.033). More interestingly, the expression of Notch-1 protein was often observed to be negative in solid predominant adenocarcinoma (SPA) tissues, but highly expressed in papillary predominant adenocarcinoma (PPA) and micropapillary predominant adenocarcinoma (MPA) tissues. Kaplan-Meier survival analysis showed that patients with positive Notch-1 expression had a prolonged progression of overall survival compared with those with negative Notch-1 expression (P = 0.033). The median survival time of Notch-1-positive or negative patients was 64.6 months (95% CI: 31.497-97.703 months) or 36.0 months (95% CI: 12.132-59.868 months).
Notch-1 could be used as a predictable biomarker to be detected in different pathological and histological subtypes in LAD for diagnosis or prognosis.
Lung adenocarcinoma; Notch-1; Immunohistochemistry; Histological subtypes; Prognosis
The purpose of this study is to evaluate the radiation dose in patients undergoing liver angiographic procedure and verify the usefulness of different dose measurements to prevent deterministic effects. Gafchromic film, MicroMOSFET data and DIAMENTOR device of the X-ray system were used to characterize the examined interventional radiology (IR) procedure.
Materials and methods
A liver embolization procedure, the SIRT (Selective Internal Radiation Therapy), was investigated. The exposure parameters from the DIAMENTOR as well as patient and geometrical data were registered. Entrance skin dose map obtained using Gafchromic film (ESDGAF) in a standard phantom as well as in 12 patients were used to calculate the maximum skin dose (MSDGAF). MicroMOSFETs were used to assess ESD in relevant points/areas. Moreover, the maximum value of five MicroMOSFETs array, due to the extension of treated area and to the relative distance of 2–3 cm of two adjacent MicroMOSFETs, was useful to predict the MSD without interfering with the clinical practice. PCXMC vers.1.5 was used to calculate effective dose (E) and equivalent dose (H).
The mean dose-area product (DAPDIAMENTOR) for SIRT procedures was 166 Gycm2, although a wide range was observed. The mean MSDGAF for SIRT procedures was 1090 mGy, although a wide range was experienced. A correlation was found between the MSDGAF measured on a patient and the DAPDIAMENTOR value for liver embolizations. MOSFET and Gafchromic data were in agreement within 5% in homogeneous area and within 20% in high dose gradient regions. The mean equivalent dose in critical organs was 89.8 mSv for kidneys, 22.9 mSv for pancreas, 20.2 mSv for small intestine and 21.0 mSv for spleen. Whereas the mean E was 3.7 mSv (range: 0.5-13.7).
Gafchromic films result useful to study patient exposure and determine localization and amplitude of high dose skin areas to better predict the skin injuries. Then, DAPDIAMENTOR or MOSFET data could offer real-time methods, as on-line dose alert, to avoid any side effects during liver embolization with prolonged duration.
Skin dose measurement; Gafchromic film dosimetry; MOSFET dosimetry; Interventional radiology; Liver embolization
Three-dimensional (3D) minimally invasive video-assisted thyroidectomy (MIVAT) was carried out with a 4-mm, 3D 0-degree stereoscopic endoscope. The procedure was applied on 3 patients who underwent total thyroidectomy and data were prospectively collected. Operative time for total thyroidectomy ranged from 72 to 90 minutes. Neither intra-nor post-operative complications were reported during the study.
The surgical team noticed a good perception of depth and easy recognising of anatomic structures, especially concerning the upper and lower vascular pedicle, the parathyroids, the superior and inferior laryngeal nerves. Preliminary impression emerging from this study seems to suggest that 3D MIVAT is safe and effective. Future studies with larger case series are required to determine the role of this procedure.
MIVAT; 3D; Three dimensional; Minimally invasive; Thyroidectomy
Nucleobindin 2 (NUCB2) protein, a novel oncoprotein, is overexpressed in breast cancer. To date, there have been no published data regarding the role of NUCB2 protein expression in prostate cancer (PCa). Therefore, this study was performed to investigate the correlations between NUCB2 protein expression and prognosis in patients with PCa.
Through immunohistochemistry, NUCB2 protein expression was evaluated in 60 benign prostatic hyperplasia (BPH) specimens and 180 PCa specimens. The correlation of NUCB2 protein expression with clinicopathological parameters was assessed using χ2 analysis. Kaplan-Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between NUCB2 protein expression and prognosis of PCa patients.
The immunohistochemistry results showed that the expression level of NUCB2 in PCa cases was significantly higher than that in BPH tissues (P < 0.001). Moreover, statistical analysis also showed that high NUCB2 protein expression was positively related to seminal vesicle invasion, lymph node metastasis, angiolymphatic invasion, higher Gleason score, biochemical recurrence (BCR), and higher preoperative prostate-specific antigen (PSA). Furthermore, it was also shown that patients with high NUCB2 protein expression had significantly poorer overall survival and BCR- free survival compared with patients with low expression of NUCB2 protein. Multivariate Cox regression analysis revealed that high NUCB2 protein expression level was an independent prognostic factor for overall survival and BCR-free survival of patients with PCa.
NUCB2 protein expression showed a strong association with the potencies of BCR and progression of PCa, and that may be applied as a novel biomarker for the prediction of BCR, and helpful for improving the diagnosis, prognosis and treatment of PCa.
Metastasis is the major cause of cancer-related death in patients with gastric cancer, and aberrant expression of various microRNAs (miRNAs) is associated with cancer metastasis.
Profiling of differentially expressed miRNAs was performed in three cases of primary gastric cancer and the corresponding metastatic lymph node tissues. Then, the five most altered miRNAs were further verified in 16 paired samples. Two of these five miRNAs were further assessed for their effects on the regulation of gastric cancer cell growth and invasion.
The miRNA profile data showed 151 upregulated miRNAs (≥ 1.5-fold) and 285 downregulated miRNAs (≤ 0.67-fold) in the metastatic tissues compared to the primary gastric cancer tissues. Among these five miRNAs (i.e., hsa-miR-508-5p, hsa-miR-30c, hsa-miR-337-3p, hsa-miR-483-5p, and hsa-miR-134), expression of hsa-miR-337-3p and hsa-miR-134 was significantly downregulated in these 16 lymph node metastatic tissues compared to their primary tumor tissues (P<0.05) and in nine gastric cancer cell lines compared to the nonmalignant GES cell line. Furthermore, induction of hsa-miR-134 or hsa-miR-337-3p expression did not dramatically affect gastric cancer cell proliferation, but transfection of the hsa-miR-337-3p mimic did reduce gastric cancer cell invasion capacity.
These findings indicate that hsa-miR-337-3p plays a role in the reduction of gastric cancer cell invasion capacity, and further studies on the mechanism of hsa-miR-337-3p in gastric cancer metastasis are warranted.
Gastric cancer; miRNA; Metastasis; hsa-miR-337-3p; miRNA array
Osteosarcoma in dogs and humans share many similarities and the dog has been described as an excellent model to study this disease. The median survival in dogs has not improved in the last 25 years. Taurolidine has been shown to be cytotoxic to canine and human osteosarcoma in vitro. The goals of this study were to determine the pharmacokinetics and safety of taurolidine in healthy dogs and the safety of taurolidine in combination with doxorubicin or carboplatin in dogs with osteosarcoma.
Two percent taurolidine was infused into six healthy dogs (150 mg/kg) over a period of two hours and blood samples were taken periodically. One dog received taurolidine with polyvinylpyrrolidone (PVP) as its carrier and later received PVP-free taurolidine as did all other dogs in this study. Serum taurolidine concentrations were determined using high-performance liquid chromatography (HPLC) online coupled to ESI-MS/MS in the multiple reaction monitoring mode. Subsequently, the same dose of taurolidine was infused to seven dogs with osteosarcoma also treated with doxorubicin or carboplatin.
Taurolidine infusion was safe in 6 healthy dogs and there were no significant side effects. Maximum taurolidine serum concentrations ranged between 229 to 646 μM. The dog that received taurolidine with PVP had an immediate allergic reaction but recovered fully after the infusion was stopped. Three additional dogs with osteosarcoma received doxorubicin and taurolidine without PVP. Toxicities included dilated cardiomyopathy, protein-losing nephropathy, renal insufficiency and vasculopathy at the injection site. One dog was switched to carboplatin instead of doxorubicin and an additional 4 dogs with osteosarcoma received taurolidine-carboplatin combination. One incidence of ototoxicity occurred with the taurolidine- carboplatin combination. Bone marrow and gastro-intestinal toxicity did not appear increased with taurolidine over doxorubicin or carboplatin alone.
Taurolidine did not substantially exacerbate bone marrow or gastro-intestinal toxicity however, it is possible that taurolidine increased other toxicities of doxorubicin and carboplatin. Administering taurolidine in combination with 30 mg/m2 doxorubicin in dogs is not recommended but taurolidine in combination with carboplatin (300 mg/m2) appears safe.
Taurolidine; Doxorubicin; Carboplatin; Osteosarcoma; Dog
Cutaneous melanoma is a malignant neoplasm with a constantly increasing incidence, the prognosis of which is largely dependent on early diagnosis. The appropriateness of requests for ultrasound (US) tests during melanoma follow-up of patients referred to our institute was evaluated.
Patients and methods
The requests for US tests of all patients referred to our institute over a four-month period were assessed. In order to correctly evaluate the appropriateness of requests, patients were split into two groups on the basis of melanoma thickness: > 1 mm (Group A) and < 1 mm (Group B).
546 patients were enrolled in our study out of a total of 1240 US tests performed. Out of 290 Group A patients, 104 patients (35%) did not meet the established congruity criteria. Group B was composed of 256 individuals, 92 patients (35.9%) of which were found to have at least one inappropriate request.
In our study, more than 30% of the requests for US tests were found to be inappropriate, to the detriment of those with a real need for diagnostic testing. This lengthens waiting lists and it may also increase public healthcare costs. Therefore, it is mandatory to adopt new, widely accepted and easily applicable guidelines.
Cutaneous melanoma; Defensive medicine; Ultrasound test appropriateness
Mutations of the p53 oncosuppressor gene are amongst the most frequent aberration seen in human cancer. Some mutant (mt) p53 proteins are prone to loss of Zn(II) ion that is bound to the wild-type (wt) core, promoting protein aggregation and therefore unfolding. Misfolded p53 protein conformation impairs wtp53-DNA binding and transactivation activities, favouring tumor growth and resistance to antitumor therapies. Screening studies, devoted to identify small molecules that reactivate mtp53, represent therefore an attractive anti-cancer therapeutic strategy. Here we tested a novel fluorescent curcumin-based Zn(II)-complex (Zn-curc) to evaluate its effect on mtp53 reactivation in cancer cells.
P53 protein conformation was examined after Zn-curc treatment by immunoprecipitation and immunofluorescence assays, using conformation-specific antibodies. The mtp53 reactivation was evaluated by chromatin-immunoprecipitation (ChIP) and semi-quantitative RT-PCR analyses of wild-type p53 target genes. The intratumoral Zn-curc localization was evaluated by immunofluorescence analysis of glioblastoma tissues of an ortothopic mice model.
The Zn-curc complex induced conformational change in p53-R175H and -R273H mutant proteins, two of the most common p53 mutations. Zn-curc treatment restored wtp53-DNA binding and transactivation functions and induced apoptotic cell death. In vivo studies showed that the Zn-curc complex reached glioblastoma tissues of an ortothopic mice model, highlighting its ability to crossed the blood-tumor barrier.
Our results demonstrate that Zn-curc complex may reactivate specific mtp53 proteins and that may cross the blood-tumor barrier, becoming a promising compound for the development of drugs to halt tumor growth.
Mutant p53; Protein conformation; p53 transcriptional activity; DNA binding; Zinc complex; Cancer therapy; Glioblastoma; Gene expression
Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model.
SUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice.
The results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo.
Our results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC.
Inflammatory breast cancer; EZH2; Cancer stem cell; Tumor spheroid formation
Recently, we reported an association of a novel cancer testis (CT) antigen, sperm-associated antigen 9 (SPAG9) expression in breast cancer clinical samples, indicating its potential role in carcinogenesis. Around 15% breast cancers are designated as triple-negative for which treatment modalities are limited. Therefore, in the present study, we assessed the role of SPAG9 in triple-negative breast cancer cells.
SPAG9 mRNA and protein expression was investigated in various breast cancer cells of different hormone receptor status and different subtypes by employing reverse transcriptase-polymerase chain reaction (RT-PCR), real time PCR, Western blotting, indirect immunofluorescence (IIF) and fluorescence activated cell sorting (FACS). Employing plasmid-based small interfering RNA (siRNA) approach, knockdown of SPAG9 was carried out in triple-negative breast cancer cells, MDA-MB-231, to assess its role on various malignant properties in vitro and in vivo.
SPAG9 mRNA and protein expression was detected in all breast cancer cells. Further, IIF results showed that SPAG9 was predominantly localized in the cytoplasm of breast cancer cells. FACS analysis revealed distinct SPAG9 surface localization in breast cancer cells. Gene silencing of SPAG9 resulted in significant reduction in cellular proliferation, colony forming ability, migration, invasion and cellular motility of MDA-MB-231 cells. Further, ablation of SPAG9 expression resulted in reduction in the tumor growth of human breast cancer xenograft in nude mice in vivo.
In summary, our data indicated that down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells, suggesting that SPAG9 may be a potential target for therapeutic use.
Migration; Invasion; CT antigens; SPAG9; Gene silencing
Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment.
The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant β2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay.
Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with β2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities.
Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment.
Telomere targeting agents; G-quadruplex; Anti-cancer therapy
The role of second-line therapy in gastric cancer patients mostly stemmed from clinical trials with monochemotherapy carried out in Asian countries. Nevertheless, these results cannot be broadly generalized as molecular studies suggested the existence of different sets of deregulated gene networks correlated with ethnicity. In the present study, we investigated the activity and safety of FOLFIRI given as a second-line therapy in metastatic gastric or gastro-esophageal junction cancer patients who experienced disease progression on or after first-line docetaxel-containing chemotherapy.
Patients with histologically confirmed metastatic gastric cancer who failed docetaxel-containing first-line therapy and who received FOLFIRI in second line were eligible for the study. Seventy patients treated at three Italian cancer centers between 2005 and 2012 entered the study. Patients received every 2 weeks irinotecan 180 mg/m2 as 1 h infusion on day 1, folinic acid 100 mg/m2 intravenously days 1–2, and fluorouracil as a 400 mg/m2 bolus and then 600 mg/m2 continuous infusion over 22 hours days 1–2.
We observed 1(1.4%) complete response, 15 (21.4%) partial response, for an overall response rate of 22.8% (95% confidence interval (CI): 13.4-32.3). Stable disease was recorded in 21 (30%) patients. Median progression-free survival and overall survival were 3.8 months (95% CI: 3.3-4.4) and 6.2 months (95% CI: 5.3-7.1), respectively. The treatment was well tolerated, as the most common G3-4 toxicities were neutropenia (28.5%) and diarrhea (14.5%).
FOLFIRI appears an effective and safe treatment option for pretreated metastatic gastric cancer patients, and deserves further investigation in randomized clinical trials.
FOLFIRI; Gastric cancer; Second-line chemotherapy
L1 cell adhesion molecule (L1CAM) and epithelial cell adhesion molecule (EPCAM) have been implicated in the development and progression of gastric cancer. The present study investigated the clinical significance of L1CAM and EPCAM in the development, progression and prognosis of gastric cancer.
Expression of L1CAM and EPCAM were examined immunochemically in 601 clinicopathologically characterized gastric cancer cases.
L1CAM protein was detected in 23.9% of human non-tumor mucosa samples. All samples expressed L1CAM protein at low levels. High expression of L1CAM protein was detected in 163 (27.1%) tumors. Expression of L1CAM correlated with age, tumor location, size of tumors, Lauren’s classification, depth of invasion, lymph node and distant metastases, regional lymph node stage, Tumor-Node-Metastasis (TNM) stage and prognosis. EPCAM protein was detected in 45.7% of human non-tumor mucosa samples. All samples expressed EPCAM protein at low levels. High expression of EPCAM protein was detected in 247 (41.1%) tumors. Expression of EPCAM correlated with age, tumor location, size of tumors, Lauren’s classification, depth of invasion, lymph node and distant metastases, regional lymph node stage, TNM stage and prognosis. Cumulative 5-year survival rates of patients with high expression of both L1CAM and EPCAM were significantly lower than in patients with low expression of both.
Expression of L1CAM and EPCAM in gastric cancer was significantly associated with lymph node and distant metastasis, and poor prognosis. L1CAM and EPCAM proteins could be useful markers to predict tumor progression and prognosis.
Gastric carcinoma; L1CAM; EPCAM; Immunohistochemistry; Progression; Prognosis
To detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis.
A surgical specimen obtained from a 68-year-old female HCC patient was analyzed by triple combination array, which identified doublecortin domain-containing 2 (DCDC2) as a candidate tumor suppressor gene of HCC. Subsequently, samples from 48 HCC patients were evaluated for their DCDC2 methylation and expression status using methylation specific PCR (MSP) and semi-quantitative reverse transcriptase (RT) PCR, respectively. Then, we investigated the relationship between clinicopathological factors and methylation status of DCDC2.
DCDC2 was revealed to be hypermethylated (methylation value 0.846, range 0–1.0) in cancer tissue, compared with adjacent normal tissue (0.212) by methylation array in the 68-year-old female patient. Expression array showed decreased expression of DCDC2 in cancerous tissue. SNP array showed that the copy number of chromosome 6p22.1, in which DCDC2 resides, was normal. MSP revealed hypermethylation of the promoter region of DCDC2 in 41 of the tumor samples. DCDC2 expression was significantly decreased in the cases with methylation (P = 0.048). Furthermore, the methylated cases revealed worse prognosis for overall survival than unmethylated cases (P = 0.048).
The present study indicates that triple combination array is an effective method to detect novel genes related to HCC. We propose that DCDC2 is a tumor suppressor gene of HCC.
DCDC2; Hepatocellular carcinoma; Methylation; Triple combination array
Among the numerous genetic defects associated with hepatocarcinogenesis, telomere abnormalities appear to play a role both in tumor promotion and maintenance. Telomeres, the chromosome extremities, are protected by specific proteins, the shelterin complex and by additional factors. Besides telomerase dysregulation, expression changes of these telomere factors have been observed in cancers.
Here, we tested the hypothesis that such dysregulation might occur in hepatocellular carcinoma (HCC) with specific patterns depending on the cause of HCC. We compared telomere length, telomerase activity (TA), hTERT and telomere genes expression using PCR and Western-blot analyses between non-cirrhotic liver, peritumoral cirrhotic tissue (40 samples) and cancerous tissue (40 samples) derived from 40 patients with HBV-, HCV-, or alcohol-related HCC.
Alterations in TA, hTERT expression and telomere length between non-cirrhotic, cirrhotic, and tumor samples were not significantly influenced by the cause of HCC. In contrast, the expression pattern of hTR, shelterin, and non-shelterin telomere protective factors clearly distinguished the 3 causes of cirrhosis and HCC. For patients with HBV diseased liver, when compared with non-cirrhotic liver, the cirrhotic tissue underexpressed all shelterin and all but HMRE11A and RAD50 non-shelterin telomere factors. For HCV the expression level of POT1, RAP1, Ku80, and RAD50 was higher in cirrhotic than in non-cirrhotic liver samples without evidence for significant transcriptional change for the remaining genes. For alcohol-related liver diseases, the expression level of POT1, RAP1, TIN2, hMRE11A, hMRE11B, Ku70, Ku80, RAD50, TANK1, and PINX1 was higher in cirrhotic than in non-cirrhotic liver samples. For the 3 causes of HCC, there was no significant change in shelterin and non-shelterin gene expression between cirrhosis and HCC samples.
These results validate our hypotheses and demonstrate that cirrhosis and HCC add-up numerous telomere dysfunctions including numerous cause-specific changes that appear to occur early during the course of the disease.
Liver; Hepatocellular carcinoma; Telomere; Telomerase; Shelterin; Hepatitis B virus; Hepatitis C virus; Alcohol; Cirrhosis
Cellfood™ (CF) is a nutritional supplement containing deuterium sulphate, minerals, amino acids, and enzymes, with well documented antioxidant properties. Its organic and inorganic components are extracted from the red algae Lithothamnion calcareum, whose mineral extract has shown growth-inhibitory effect both on in vitro and in vivo models. The purpose of this study was to evaluate the antiproliferative effects of CF on leukemic cells. In fact, according to its capacity to modulate O2 availability and to improve mitochondrial respiratory metabolism, we wondered if CF could affect cancer cell metabolism making cells susceptible to apoptosis.
Three leukemic cell lines, Jurkat, U937, and K562, were treated with CF 5 μl/ml up to 72 hours. Cell viability, apoptosis (i.e. caspase-3 activity and DNA fragmentation), hypoxia inducible factor 1 alpha (HIF-1α) concentration, glucose transporter 1 (GLUT-1) expression, lactate dehydrogenase (LDH) activity and lactate release in the culture medium were detected and compared with untreated cells.
CF significantly inhibited leukemic cell viability by promoting cell apoptosis, as revealed by caspase-3 activation and DNA laddering. In particular, CF treated cells showed lower HIF-1α levels and lower GLUT-1 expression as compared to untreated cells. At the same time, CF was able to reduce LDH activity and, consequently, the amount of lactate released in the extracellular environment.
We supplied evidence for an antiproliferative effect of CF on leukemia cell lines by inducing cell death through an apoptotic mechanism and by altering cancer cell metabolism through HIF-1α and GLUT-1 regulation. Thanks to its antioxidative and proapoptotic properties, CF might be a good candidate for cancer prevention.
Apoptosis; Cellfood™; Glucose transporter 1; Hypoxia inducible factor 1 alpha; Lactate dehydrogenase; Leukemic cells; Tumor metabolism
Increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT), which occurs during cancer progression and metastasis. Recent studies have indicated the expression of receptor activator of nuclear factor-κB (RANK) in various solid tumors, including breast cancer. Although activation of the RANK ligand (RANKL)/RANK system promotes cell migration, metastasis, and anchorage-independent growth of tumor-initiating cells, it remains to be investigated if RANKL induces EMT in breast cancer cells. In this study, we investigated whether RANKL induces EMT in normal breast mammary epithelial cells and breast cancer cells, and the mechanism underlying such induction.
Expression levels of vimentin, N-cadherin, E-cadherin, Snail, Slug, and Twist were examined by real-time polymerase chain reaction. Cell migration and invasion were assessed using Boyden chamber and invasion assays, respectively. The effects of RANKL on signal transduction molecules were determined by western blot analyses.
We found that stimulation by RANKL altered the cell morphology to the mesenchymal phenotype in normal breast epithelial and breast cancer cells. In addition, RANKL increased the expression levels of vimentin, N-cadherin, Snail, and Twist and decreased the expression of E-cadherin. We also found that RANKL activated nuclear factor-κB (NF-κB), but not extracellular signal-regulated kinase 1/2, Akt, mammalian target of rapamycin, c-Jun N-terminal kinase, and signal transducer and activator of transcription 3. Moreover, dimethyl fumarate, a NF-κB inhibitor, inhibited RANKL-induced EMT, cell migration, and invasion, and upregulated the expressions of Snail, Twist, vimentin, and N-cadherin.
The results indicate that RANKL induces EMT by activating the NF-κB pathway and enhancing Snail and Twist expression. These findings suggest that the RANKL/RANK system promotes tumor cell migration, invasion, and metastasis via the induction of EMT.
RANK; RANKL; EMT; Breast cancer; NF-κB
There is no consensus regarding the secondary cytoreduction surgery (CRS) in recurrent ovarian cancer patients. The present study aims to determine the value of secondary CRS and the eligible subgroup for this procedure.
96 platinum-sensitive recurrent ovarian cancer patients were recruited from Jiangsu Institute of Cancer Research between 1992 and 2011. Follow-up was conducted based on the surveillance protocol of MD Anderson Cancer Center. Cox proportional hazards model and log-rank test were used to assess the associations between the survival durations and covariates. Logistic regression analysis was used to explore optimal secondary CRS related factors.
Optimal secondary CRS was associated with time to progression (TTP) and overall survival (OS) in patients (p < 0.01 both). Optimal secondary CRS and asymptomatic recurrent were similarly associated with longer OS (median: 79.2 vs. 53.9 and 76.1 vs. 56.0 months with p = 0.02 and p = 0.04, respectively) and TTP (median: 13.9 vs. 10.5 and 19.3 vs. 9.0 months with p = 0.02 and p = 0.03, respectively) than counterparts. Optimal initial CRS (p = 0.01), asymptomatic recurrent (p = 0.02) and longer progression-free survival duration (p = 0.02) were the independent indicators of optimal secondary CRS.
Optimal secondary CRS had survival benefit for platinum-sensitive epithelial ovarian cancer. Asymptomatic recurrent was one of the recruited factors for this procedure.
Epithelial ovarian cancer; CA-125; Clinical relapse; Cytoreductive surgery; Time to progression
The deleted in liver cancer 1 (DLC1) and plasminogen activator inhibitor 1 (PAI-1) are known to be closely associated with tumor growth and metastasis in several kinds of human tumors. The aim of this study was to investigate the expression of DLC1 and PAI-1 in ovarian carcinoma, and evaluate their relations with the prognosis of ovarian carcinoma.
Immunohistochemical staining and Western blot were used to examine the expressions of DLC1 and PAI-1 protein in 25 specimens normal ovarian tissues, 52 specimens of serous cystadenocarcinoma tissues and 23 specimens of mucinous cystadenocarcinoma tissues. Chi-square test, Logistic regression and Partial Correlate analysis were performed to evaluate the association between DLC1 and PAI-1 with clinicopathological characteristics. Overall survival was estimated by Kaplan-Meier curves and multivariate Cox analysis. The relationships between DLC1 and PAI-1 protein expression were analyzed by Pearson’s correlation coefficient.
The expression of DLC1 protein in ovarian carcinoma tissues was significantly lower than that in normal ovarian tissues, but it was converse for PAI-1. In ovarian carcinoma, the expression of DLC1 was significantly associated with advanced FIGO stage, ascites and positive lymph node metastasis, whereas PAI-1 protein was closely related with advanced FIGO stage, poor histological differentiation and lymph node metastasis. The expression of DLC1 was negatively correlated with PAI-1 in ovarian carcinoma. Ovarian cancer patients with negative expression of DLC1 and positive expression of PAI-1 had the worst overall survival time compared to other patients.
The expression of DLC1 and PAI-1 were closely related with the metastasis and invasion of ovarian carcinoma, only the combination of DLC1 and PAI-1 could serve as an independent prognostic factor of ovarian carcinoma.
Ovarian cancer; Deleted in liver cancer 1; Plasminogen activator inhibitor 1; Invasion; Metastasis; Prognosis
To date, no prognostic microRNAs (miRNAs) for isocitrate dehydrogenase 1 (IDH1) wild-type glioblastoma multiformes (GBM) have been reported. The aim of the present study was to identify a miRNA signature of prognostic value for IDH1 wild-type GBM patients using miRNA expression dataset from the The Cancer Genome Atlas (TCGA).
Differential expression profiling analysis of miRNAs was performed on samples from 187 GBM patients, comprising 17 mutant-type IDH1 and 170 wild-type IDH1 samples.
A 23-micoRNA signature which was specific to the IDH1 mutation was revealed. Survival data was available for 140 of the GBM patients with wild-type IDH1. Using these data, the samples were characterized as high-risk or low-risk group according to the ranked protective scores for each of the 23 miRNAs in the 23-miRNA signature. Then, the 23 IDH1 mutation-specific miRNAs were classified as risky group and protective group miRNAs based on the significance analysis of microarrays d-score (SAM d-value) (positive value or negative value). The risky group miRNAs were found to be expressed more in the high-risk samples while the protective group miRNAs were expressed more in the low-risk samples. Patients with high protective scores had longer survival times than those with low protective scores.
These findings show that IDH1 mutation-specific miRNA signature is a marker for favorable prognosis in primary GBM patients with the IDH1 wild type.
IDH1; Wild type; MiRNA signature; Glioblastoma
An External Quality Assessment (EQA) program was developed to investigate the state of the art of HER2 immunohistochemical determination in breast cancer (BC) in 16 Pathology Departments in the Lazio Region (Italy). This program was implemented through two specific steps to evaluate HER2 staining (step 1) and interpretation (step 2) reproducibility among participants.
The management activities of this EQA program were assigned to the Coordinating Center (CC), the Revising Centers (RCs) and the Participating Centers (PCs). In step 1, 4 BC sections, selected by RCs, were stained by each PC using their own procedures. In step 2, each PC interpreted HER2 score in 10 BC sections stained by the CC. The concordance pattern was evaluated by using the kappa category-specific statistic and/or the weighted kappa statistic with the corresponding 95% Jackknife confidence interval.
In step 1, a substantial/almost perfect agreement was reached between the PCs for scores 0 and 3+ whereas a moderate and fair agreement was observed for scores 1+ and 2+, respectively.
In step 2, a fully satisfactory agreement was observed for 6 out of the 16 PCs and a quite satisfactory agreement was obtained for the remaining 10 PCs.
Our findings highlight that in the whole HER2 evaluation process the two intermediate categories, scores 1+ and 2+, are less reproducible than scores 0 and 3+. These findings are relevant in clinical practice where the choice of treatment is based on HER2 positivity, suggesting the need to share evaluation procedures within laboratories and implement educational programs.
Breast Neoplasm/drug therapy; Receptor erbB-2/analysis; Immunohistochemistry/methods; Quality control; Reproducibility of results
Renal cell carcinoma (RCC), the most common malignancy of the kidney, is refractory to standard therapy and has an incidence that continues to rise. Screening of plant extracts in search of new agents to treat RCC resulted in the discovery of englerin A (EA), a natural product exhibiting potent selective cytotoxicity against renal cancer cells. Despite the establishment of synthetic routes to the synthesis of EA, very little is known about its mechanism of action. The results of the current study demonstrate for the first time that EA induces apoptosis in A498 renal cancer cells in addition to necrosis. The induction of apoptosis by EA required at least 24 h and was caspase independent. In addition, EA induced increased levels of autophagic vesicles in A498 cells which could be inhibited by nonessential amino acids (NEAA), known inhibitors of autophagy. Interestingly, inhibition of autophagy by NEAA did not diminish cell death suggesting that autophagy is not a cell death mechanism and likely represents a cell survival mechanism which ultimately fails. Apart from cell death, our results demonstrated that cells treated with EA accumulated in the G2 phase of the cell cycle indicating a block in G2/M transition. Moreover, our results determined that EA inhibited the activation of both AKT and ERK, kinases which are activated in cancer and implicated in unrestricted cell proliferation and induction of autophagy. The phosphorylation status of the cellular energy sensor, AMPK, appeared unaffected by EA. The high renal cancer selectivity of EA combined with its ability to induce multiple mechanisms of cell death while inhibiting pathways driving cell proliferation, suggest that EA is a highly unique agent with great potential as a therapeutic lead for the treatment of RCC.
Englerin A; Apoptosis; Autophagy; Necrosis; Renal cell carcinoma