Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with dismal prognosis and high incidence and mortality in Kazakh population. MiR-34a, a direct p53 target gene, possesses tumor-suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. The reduced expression of miR-34a by methylation in various cancers has been reported.
To determine whether aberrant miR-34a methylation occurs in esophageal cancer, the DNA methylation of 23 CpGs sites in the miR-34a promoter was quantitatively analyzed in relation to the translation initiation site by MALDI -TOF mass spectrometry in 59 ESCC tissues and 34 normal tissues from the Kazakh population. Real-time PCR was used to detect the inhibition of miR-34a expression levels and to evaluate their association with methylation.
We found that miR-34a is more frequently methylated in ESCC (0.133 ± 0.040) than in controls (0.066 ± 0.045, P < 0.01). A nearly two-fold increase in miR-34a expression for the hypomethylated promoter was found in normal esophageal tissues than ESCC with hypermethylation (P <0.0001), pointing to a negative relationship between miR-34a CpG sites methylation and expression(r = −0.594, P = 0.042). The hypermethylation of miR-34a CpG_8.9 was associated with the advanced UICC stage III/IV of the esophageal cancers, and the hypermethylation of CpG_8.9 and CpG_5 of miR-34a was significantly correlated with lymph node metastasis.
Our findings suggest that miR-34a is involved in the etiology of ESCC and that hypermethylated miR-34a is a potential biomarker for ESCC diagnosis and prognosis. Moreover, targeting miR-34a methylation by demethylating agents may offer a novel strategy for anticancer therapy of ESCC.
MiR-34a; Esophageal squamous cell carcinoma; Kazakh; Methylation
The chaperon heat shock protein 90 (HSP90) constitutes an important target for anti-tumor therapy due to its essential role in the stabilization of oncogenes. However, HSP90 is ubiquitously active to orchestrate protein turnover, chemotherapeutics that target HSP90 may affect immune cells as a significant side effect. Therefore, we asked for potential effects of pharmacological HSP90 inhibition at a therapeutically relevant concentration on human dendritic cells (DCs) as main inducers of both cellular and humoral immune responses, and on human CD4+ T cells as directly activated by DCs and essential to confer B cell help.
Unstimulated human monocyte-derived DCs (MO-DCs) were treated with the prototypical HSP90 inhibitor geldanamycin (GA). Based on dose titration studies performed to assess cytotoxic effects, GA was applied at a rather low concentration, comparable to serum levels of clinically used HSP90 inhibitors. The immuno-phenotype (surface markers, cytokines), migratory capacity, allo T cell stimulatory and polarizing properties (proliferation, cytokine pattern) of GA-treated MO-DCs were assessed. Moreover, effects of GA on resting and differentially stimulated CD4+ T cells in terms of cytotoxicity and proliferation were analysed.
GA induced partial activation of unstimulated MO-DCs. In contrast, when coapplied in the course of MO-DC stimulation, GA prevented the acquisition of a fully mature DC phenotype. Consequently, this MO-DC population exerted lower allo CD4+ T cell stimulation and cytokine production. Furthermore, GA exerted no cytotoxic effect on resting T cells, but abrogated proliferation of T cells stimulated by MO-DCs at either state of activation or by stimulatory antibodies.
HSP90 inhibitors at clinically relevant concentrations may modulate adaptive immune responses both on the level of DC activation and T cell proliferation. Surprisingly, unstimulated DCs may be partially activated by that agent. However, due to the potent detrimental effects of HSP90 inhibitors on stimulated CD4+ T cells, as an outcome a patients T cell responses might be impaired. Therefore, HSP90 inhibitors most probably are not suitable for treatment in combination with immunotherapeutic approaches aimed to induce DC/T cell activation.
Heat shock protein 90; Geldanamycin; NF-κB; RelB; Dendritic cell; T cell
Over the last decades, billions have been spent and huge efforts have been taken in basic and clinical cancer research [CA Cancer J Clin
11-30]. About a decade ago, the arms race between drugs and cancer cells reached a new level by introduction of tyrosine kinase inhibitors (TKI) into pharmacological anti-cancer therapy. According to their molecular mechanism of action, TKI in contrast to so-called “classic” or “conventional” cytostatics belong to the group of targeted cancer medicines, characterized by accurately fitting with biological structures (i.e. active centers of kinases). Numerous (partly orphan) indications are covered by this new class of substances. Approximately ten years after the first substances of this class of medicines were authorized, patent protection will end within the next years. The following article covers clinical meaning and regulatory status of anti-cancer TKI and gives an outlook to what is expected from the introduction of generic anti-cancer TKI.
Narrow therapeutic index drugs (NTID); Tyrosine kinase inhibitors (TKI); Orphan drug status
Cholangiocarcinoma (CCA) is highly resistant to most of the known chemotherapeutic treatments. NAD(P)H-quinone oxidoreductase 1 (NQO1) is an antioxidant/detoxifying enzyme recently recognized as an important contributor to chemoresistance in some human cancers. However, the contribution of NQO1 to chemotherapy resistance in CCA is unknown.
Two CCA cell lines, KKU-100 and KKU-M214, with high and low NQO1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents; 5-fluorouracil (5-FU), doxorubicin (Doxo), and gemcitabine (Gem). NQO1 and/or p53 expression in KKU-100 cells were knocked down by siRNA. NQO1 was over-expressed in KKU-M214 cells by transfection with pCMV6-XL5-NQO1 expression vector. CCA cells with modulated NQO1 and/or p53 expression were treated with chemotherapeutic agents, and the cytotoxicity was assessed by SRB assay. The mechanism of enhanced chemosensitivity was evaluated by Western blot analysis.
When NQO1 was knocked down, KKU-100 cells became more susceptible to all chemotherapeutic agents. Conversely, with over-expression of NQO1 made KKU-M214 cells more resistant to chemotherapeutic agents. Western blot analysis suggested that enhanced chemosensitivity was probably due to the activation of p53-mediated cell death. Enhanced susceptibility to chemotherapeutic agents by NQO1 silencing was abolished by knockdown of p53.
These results suggest that inhibition of NQO1 could enhance the susceptibility of CCA to an array of chemotherapeutic agents.
NAD(P)H-quinone oxidoreductase 1 (NQO1); Cholangiocarcinoma (CCA); 5-fluorouracil (5-FU); Doxorubicin (Doxo); Gemcitabine (Gem); p53
ErbB4 expression has been noted in various tumors, but its regulatory mechanism in esophageal squamous cell carcinoma (ESCC) remains unclear. The aim of this study was to investigate whether miR-302b regulates the expression of ErbB4 at the post-transcriptional level and to determine its expression, significance, and function in ESCC.
We used real-time reverse transcriptase-polymerase chain reaction to quantify the expression of miR-302b in 50 ESCC tissues and analyzed its relationship with clinicopathological factors and survival. Then, we investigated the post-transcriptional regulation of ErbB4 expression using immunoblot analysis and luciferase reporter assays. Finally, the effects of miR-302b on proliferation, apoptosis, and invasion of ESCC cells was detected using MTT, flow cytometric analysis, and transwell invasion assays, respectively.
miR-302b was significantly down-regulated and correlated with tumor differentiation and lymph node metastasis in ESCC. Univariate and multivariate analyses indicated that low miR-302b expression might be a poor prognostic factor. Further studies demonstrated that miR-302b post-transcriptionally down-regulated the expression of ErbB4 in vitro. Moreover, miR-302b inhibited proliferation by inducing apoptosis and repressed invasion in the ESCC cell lines.
miR-302b is a potential molecular marker of ESCC and functions as a tumor suppressor by post-transcriptionally regulating ErbB4.
miR-302b; ErbB4; Esophageal squamous cell carcinoma
c-Jun NH2-terminal kinases (JNKs) are strongly activated by a stressful cellular environment, such as chemotherapy and oxidative stress. Autophagy is a protein-degradation system in which double-membrane vacuoles called autophagosomes are formed. The autophagy-related gene Beclin 1 plays a key role in this process. We previously found that autophagy was induced by dihydroartemisinin (DHA) in pancreatic cancer cells. However, little is known about the complex relationship between ROS, JNK activation, autophagy induction, and Beclin 1 expression.
Cell viability and CCK-8 assays were carried out to determine the cell proliferation; small interfering RNAs (siRNAs) were used to knockdown c-Jun NH2-terminal kinases (JNK1/2) genes; western blot was performed to detect the protein expression of LC3, JNK, Beclin 1, caspase 3 and β-actin; production of intracellular ROS was analyzed using FACS flow cytometry; autophagy induction was confirmed by electron microscopy.
In the present study, we explored the role of DHA and Beclin 1 expression in autophagy. DHA-treated cells showed autophagy characteristics, and DHA also activated the JNK pathway and up-regulated the expression of Beclin 1. Conversely, blocking JNK signaling inhibited Beclin 1 up-regulation. JNK activation was found to primarily depend on reactive oxygen species (ROS) resulting from the DHA treatment. Moreover, JNK pathway inhibition and Beclin 1 silencing prevented the induction of DHA-induced autophagy.
These results suggest that the induction of autophagy by DHA is required for JNK-mediated Beclin 1 expression.
c-Jun NH2-terminal kinase; Beclin 1; Apoptosis; LC3; Autophagy; Pancreatic cancer; Dihydroartemisinin
The mechanism for inactivation of positive regulatory domain containing I (PRDM1), a newly identified tumour suppressor gene in extranodal NK/T-cell lymphoma, nasal type (EN-NK/T-NT) has not been well defined. The aim of the present study was to investigate the expression of PRDM1 in EN-NK/T-NT and analyse its downregulation by miRNAs.
PRDM1 and miRNA expression were evaluated in EN-NK/T-NT samples by immunohistochemical analysis, qRT-PCR, and in situ hybridisation. Luciferase assays were performed to verify the direct binding of miR-223 to the 3′-untranslated region of PRDM1 mRNA. In addition, the effect of miR-223 on PRDM1 expression was assessed in NK/T lymphoma cell lines by transfecting a miR-223 mimic or inhibitor to increase or decrease the effective expression of miR-223. Overall survival and failure-free survival in EN-NK/T-NT patients were analysed using Kaplan-Meier single-factor analysis and the log-rank test.
Investigation of the downregulation of PRDM1 in EN-NK/T-NT cases revealed that PRDM1-positive staining might be a favourable predictor of overall survival and failure-free survival in EN-NK/T-NT patients. However, the negative staining of PRDM1 usually presented transcripts, suggesting a possible post-transcriptional regulation. miR-223 and its putative target gene, PRDM1, exhibited opposite patterns of expression in EN-NK/T-NT tissues and cell lines. Moreover, PRDM1 was identified as a direct target gene of miR-223 by luciferase assays. The ectopic expression of miR-223 led to the downregulation of the PRDM1 protein in the NK/T-cell lymphoma cell line, whereas a decrease in miR-223 restored the level of PRDM1 protein.
Our findings reveal that the downregulation of the tumour suppressor PRDM1 in EN-NK/T-NT samples is mediated by miR-223 and that PRDM1-positive staining might have prognostic value for evaluating the clinical outcome of EN-NK/T-NT patients.
Extranodal NK/T-cell lymphoma; Nasal type; PRDM1; miR-223
Hec1 (NDC80) is an integral part of the kinetochore and is overexpressed in a variety of human cancers, making it an attractive molecular target for the design of novel anticancer therapeutics. A highly potent first-in-class compound targeting Hec1, TAI-1, was identified and is characterized in this study to determine its potential as an anticancer agent for clinical utility.
The in vitro potency, cancer cell specificity, synergy activity, and markers for response of TAI-1 were evaluated with cell lines. Mechanism of action was confirmed with western blotting and immunofluorescent staining. The in vivo potency of TAI-1 was evaluated in three xenograft models in mice. Preliminary toxicity was evaluated in mice. Specificity to the target was tested with a kinase panel. Cardiac safety was evaluated with hERG assay. Clinical correlation was performed with human gene database.
TAI-1 showed strong potency across a broad spectrum of tumor cells. TAI-1 disrupted Hec1-Nek2 protein interaction, led to Nek2 degradation, induced significant chromosomal misalignment in metaphase, and induced apoptotic cell death. TAI-1 was effective orally in in vivo animal models of triple negative breast cancer, colon cancer and liver cancer. Preliminary toxicity shows no effect on the body weights, organ weights, and blood indices at efficacious doses. TAI-1 shows high specificity to cancer cells and to target and had no effect on the cardiac channel hERG. TAI-1 is synergistic with doxorubicin, topotecan and paclitaxel in leukemia, breast and liver cancer cells. Sensitivity to TAI-1 was associated with the status of RB and P53 gene. Knockdown of RB and P53 in cancer cells increased sensitivity to TAI-1. Hec1-overexpressing molecular subtypes of human lung cancer were identified.
The excellent potency, safety and synergistic profiles of this potent first-in-class Hec1-targeted small molecule TAI-1 show its potential for clinically utility in anti-cancer treatment regimens.
Hec1; NDC80; Anti-cancer drug; Therapeutics; Mitosis; Apoptosis; P53; Retinoblastoma gene; Markers for response
EGFR mutation detection has been widely applied in the prediction of TKIs therapy in Non-Small Cell Lung Cancer (NSCLC). Metastatic tumors rather than primary tumors were usually assayed for those patients in advanced stages. Although the difference of EGFR mutation status in primary and metastatic tumors has been reported, the quantitative difference (ratio of mutated EGFR among total EGFR) in primary and metastatic tumors as well as in different sites of primary tumors was not clear.
Genomic DNA in Formalin Fixed-Paraffin Embedded samples of primary and metastatic tumors of 50 NSCLC patients was extracted. Real-time fluorescent PCR was performed to quantify the EGFR mutation ratios.
The EGFR mutation ratios detected in different sites of primary tumors were highly concordant, whereas the EGFR mutation ratios in metastatic tumors were lower than those in primary tumors.
Randomly chosen sample may reliably represent the type and ratio of mutations of EGFR in primary tumors. EGFR mutation ratios in primary tumors and metastatic tumors are different. If metastatic tumors are used for the detection of EGFR mutation, the sensitivity of the detection assay must be considered.
Non-small cell lung cancer; EGFR mutations; Primary tumors; Metastatic tumors; Quantification
Parthenolide (PTL) is a sesquiterpene lactone which can induce apoptosis in cancer cells and eradicate cancer stem cells such as leukemia stem cells, prostate tumor-initiating cells and so on. However, the mechanism remains largely unclear.
Lung cancer cells were treated with parthenolide and the cell lysates were prepared to detect the given proteins by Western Blot analysis, and the cell survival was assayed by SRB and MTT assay. Cell cycle was evaluated by DNA flow cytometry analysis. TNFRSF10B, PMAIP1, ATF4 and DDIT3 genes were knocked down by siRNA technique. Apoptosis was evaluated by using Annexin V-FITC/PI staining and flow cytometry analysis.
Parthenolide (PTL) induces apoptosis and cell cycle arrest in human lung cancer cells. Moreover, PTL treatment in NSCLC cells increases expression of TNFRSF10B/DR5 and PMAIP1/NOXA. Silencing of TNFRSF10B or PMAIP1 or overexpression of CFLAR /c-FLIP (long form) could protect cells from PTL-induced apoptosis. Furthermore, PTL could increase the levels of endoplasmic reticulum stress hallmarks such as ERN1, HSPA5, p-EIF2A, ATF4 and DDIT3. Knockdown of ATF4 and DDIT3 abrogated PTL-induced apoptosis, which suggested that PTL induced apoptosis in NSCLC cells through activation of endoplasmic reticulum stress pathway. More importantly, we found that ATF4, DDIT3, TNFRSF10B and PMAIP1 were up-regulated more intensively, while CFLAR and MCL1 were down-regulated more dramatically by PTL in A549/shCDH1 cells than that in control cells, suggesting that PTL preferred to kill cancer stem cell-like cells by activating more intensive ER stress response in cancer stem cell-like cells.
We showed that parthenolide not only triggered extrinsic apoptosis by up-regulating TNFRSF10B and down-regulating CFLAR, but also induced intrinsic apoptosis through increasing the expression of PMAIP1 and decreasing the level of MCL1 in NSCLC cells. In addition, parthenolide triggered stronger ER stress response in cancer stem cell-like cells which leads to its preference in apoptotic induction. In summary, PTL induces apoptosis in NSCLC cells by activating endoplasmic reticulum stress response.
Parthenolide; TNFRSF10B; CFLAR; PMAIP1; Endoplasmic reticulum stress; DDIT3
Colorectal cancer (CRC) arises as a consequence of genetic events such as gene mutation and epigenetic alteration. The aim of this study was to identify new hypermethylated candidate genes and methylation-based therapeutic targets using vincristine in CRC.
We analyzed the methylation status of 27,578 CpG sites spanning more than 14,000 genes in CRC tissues compared with adjacent normal tissues and normal colon tissues using Illumina bead chip array. Twenty-one hypermethylated genes and 18 CpG island methylator phenotype markers were selected as candidate genes. The methylation status of 39 genes was validated by quantitative methylation-specific polymerase chain reaction in CRC tissues, adjacent normal tissues, normal colon cells, and three CRC cell lines. Of these, 29 hypermethylated candidate genes were investigated using the demethylating effects of 5-aza-2′-deoxycytidine (5-aza-dC) and vincristine in CRC cells.
Thirty-two out of 39 genes were hypermethylated in CRC tissues compared with adjacent normal tissues. Vincristine induced demethylation of methylated genes in CRC cells to the same extent as 5-aza-dC. The mRNA expression of AKR1B1, CHST10, ELOVL4, FLI1, SOX5, STK33, and ZNF304 was restored by treatment with 5-aza-dC and vincristine.
These results suggest that these novel hypermethylated genes AKR1B1, CHST10, ELOVL4, SOX5, STK33, and ZNF304 may be potential methylation biomarkers and therapeutic targets of vincristine in CRC.
Colorectal cancer; Hypermethylated genes; Therapeutic targets; CIMP markers; 5-aza-2′-deoxycytidine; Vincristine
Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with 99mTc pertechnetate scintigraphy and 124I positron emission tomography (PET).
GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. 99mTc pertechnetate scintigraphy and 124I microPET imaging were performed.
GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK- 45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by 99mTc pertechnetate scintigraphy and 124I microPET imaging 2 days after treatment.
GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings.
Oncolytic viral therapy; GLV-1 h153; Gastric cancer; Human sodium iodide symporter (hNIS)
Several studies evaluated the prevalence of Lynch Syndrome (LS) in young onset colorectal cancer (CRC) patients and the results were extremely variable (5%-20%). Immunohistochemistry (IHC) for MMR proteins and/or MSI analysis are screening tests that are done, either by themselves or in conjunction, on colon cancer tissue to identify individuals at risk for LS. The primary aim of our study was to evaluate the prevalence of LS in a large series of early-onset CRC without family history compared with those with family history. The secondary aim was to assess the diagnostic accuracy of IHC and MSI analysis as pre-screening tools for LS.
Early-onset CRC patients (≤ 50 years) were prospectively recruited in the study. IHC and MSI analysis were performed in all the patients. Germ-line mutation analysis (GMA) was carried out in all MMR deficient tumors. A logistic regression model was performed to identify clinical features predictive of MSI-H.
117 early onset CRC cases were categorized in three groups (A, B, C) according with family history of CRC. IHC and MSI analysis showed MMR deficiency in 6/70 patients (8.6%) of group A, 24/40 patients (60%) of group B and none of group C. GMA showed a deleterious mutation in 19 (47.5%) patients of group B. MSI analysis had a diagnostic accuracy of 95.7% (CI 92.1-99.4) and IHC of 83.8% (CI 77.1-90.4). The logistic regression model revealed that by using a combination of the two features “No Amsterdam Criteria” and ”left sided CRC” to exclude MSI-H, accuracy was 89.7% (84.2-95.2).
Early-onset CRC patients, with left sided CRC and without family history are “at very low risk” for Lynch syndrome. The two simple criteria of family history and CRC site could be used as a pre-screening tool to evaluate whether or not patients should undergo tissue molecular screening. In the few cases of suspected LS (right sided CRC and/or Amsterdam Criteria), a reasonable approach could be to perform MSI analysis first and IHC afterwards only in MSI-H patients.
Early-onset colorectal cancer; Lynch syndrome; Immunohistochemistry; Microsatellite instability
To assess the efficacy of intraoperative ultrasound-guided implantation of 125I seeds for the treatment of unresectable pancreatic carcinoma, and analyze the associated prognostic factors.
Twenty-eight patients with pancreatic carcinoma who underwent laparotomy and were considered to have unresectable tumors were included in this study. Nine patients were pathologically diagnosed with Stage II disease, and nineteen patients with Stage III disease. Twenty-eight patients received intraoperative ultrasound-guided 125I seed implantation and received a D90 (at least 90% of the tumor volume received the reference dose) ranging from 60 to 163 Gy, with a median of 120 Gy. Seven patients received an additional 35–50 Gy external beam radiotherapy after seed implantation, and ten patients received two to ten cycles of chemotherapy. Overall survival of the patients was calculated and prognostic factors were evaluated.
Of the patients, 94.1% (16/17) achieved good to medium pain relief. The tumor response rate was 78.6% (22/28), and local control was achieved in 85.7% (24/28) of patients. The 1-, 2- and 3-year survival rates were 30%, 11% and 4%, and the median survival was 10.1 months (95% CI: 9.0-10.9). Analysis using the Cox proportional hazards model suggested that patients younger than 60 years and patients who received a D90 higher than 110 Gy may survive for a longer period.
I seed implantation provides a safe and effective method to relieve pain, control local tumor growth and, to some extent, prolong the survival of patients with stage II and III pancreatic disease, without additional complications. Age and accumulated dose may be factors predictive of a favorable outcome for patients with unresectable pancreatic carcinoma treated with 125I seeds. These findings need to be validated by conducting further studies with larger cohorts.
125I seed; Intraoperative implantation; Ultrasound-guided; Unresectable; Pancreatic carcinoma
Numerous studies have yielded inconclusive results regarding the relationship between anti-apoptotic protein Bcl-2 expression and the sensitivity to chemotherapy in the patients with breast cancer. The purpose of the current study was therefore to elaborate their relationship.
A total of 23 previously published eligible studies involving 2,467 cases were identified and included in this meta-analysis. Negative Bcl-2 expression was associated with good chemotherapy response in breast cancer patients (total objective response [OR]: risk ratio [RR] = 1.16, 95% confidence interval [CI] = 1.02-1.32, p = 0.026; total complete response [CR]: RR = 1.67, 95% CI = 1.24-2.24, p = 0.001; pathological CR: RR = 1.92, 95% CI = 1.38-2.69, p < 0.001). In further stratified analyses, this association remained for sub-groups of response in neoadjuvant chemotherapy setting, especially pathological CR. Besides, negative Bcl-2 expression was significantly associated with good OR and pathological CR in anthracycline-based chemotherapy subgroup. Furthermore, there were significant links between negative Bcl-2 expression and taxane-based chemotherapy with pathological CR, but not OR.
The results of the present meta-analysis suggest that Bcl-2 expression is a predictive factor for chemotherapy sensitivity in breast cancer patients. They could also potentially benefit further clinical treatment for breast cancers.
Bcl-2; Breast cancer; Chemotherapy; Response
To investigate the feasibility of dose escalation (86 Gy at 2 Gy/fraction) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer without androgen deprivation therapy.
Patients with histologically proven adenocarcinoma of the prostate, intermediate prognostic category, were enrolled in this study. Early and late toxicity were scored according to the Cancer Therapy Evaluation Program, Common Terminology Criteria for Adverse Events, Version 3.0. Treatment outcome was stated in terms of biochemical failure, biopsy result and clinical failure.
39 patients with a median follow-up of 71 months were analyzed. No patient experienced G3 or G4 acute gastrointestinal (GI) or genitourinary (GU) toxicity. G2 acute GI and GU toxicity were observed in 17 (44%) and 20 (51%) patients, respectively. Fourteen patients (36%) did not experience acute GI toxicity and 4 patients (10%) did not experience acute GU toxicity. G2 late GI bleeding occurred in 7 of 39 patients (18%). Both G3 and G4 late GI toxicity were seen only in one patient (2.5%). Two patients (5%) experienced G2 late GU toxicity, while G3 late GU toxicity occurred in 3 patients (8%). The 5-year actuarial freedom from biochemical failure (FFBF) was 87%. Thirty-four patients (87%) did not show biochemical relapse. Seventeen patients (44%) underwent biopsy two year after radiotherapy; of these only two were non-negative and both did not show evidence of biochemical disease.
IMRT treatment of patients with localized intermediate-risk prostate cancer at high dose levels without using androgen deprivation therapy (ADT) seems to give good disease control. Nevertheless, future trials should aim at further decreasing toxicity by exploiting image guidance techniques and by reducing the dose delivered at the interface between organs at risk and prostate.
Prostate cancer; Intermediate risk; Dose escalation; Absence of ADT; Toxicity; Outcome
Recently, leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1), a negative regulator of EGFR, was discovered is a novel agent for suppressing bladder cancer. The aim of this study was to investigate the impact of LRIG1 on the biological features of aggressive bladder cancer cells and the possible mechanisms of enhanced apoptosis induced by upregulation of LRIG1.
In this study, we examined the mRNA and protein expression of LRIG1 and EGFR in bladder cancers and normal bladder. Meanwhile, we overexpressed LRIG1 with adenovirus vector in T24/5637 bladder cancer cell lines, and we used real time-PCR, western blot, and co-immunoprecipitation analysis in order to examine the effects of LRIG1 gene on EGFR. Furthermore, we evaluate the impact of LRIG1 gene on the function of human bladder cancer cells and EGFR signaling.
The expression of LRIG1 was decreased, while the expression of EGFR was increased in the majority of bladder cancer, and the ratio of EGFR/LRIG1 was increased in tumors versus normal tissue. We found that upregulation of LRIG1 induced cell apoptosis and cell growth inhibition, and further reversed invasion in bladder cancer cell lines in vitro by inhibiting phosphorylation of downstream MAPK and AKT signaling pathway.
Taken together, our findings provide us with an insight into LRIG1 function, and we conclude that LRIG1 evolved in bladder cancer as a rare feedback negative attenuator of EGFR, thus could offer a novel therapeutic target to treat patients with bladder cancer.
LRIG1; EGFR; Apoptosis; Invasion; Bladder cancer
Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. The present treatment including surgery, chemotherapy and radiation, which have only 40% long-term cure rates, and usually cause tumor recurrence. Thus, looking for new effective and less toxic therapies has important significance. XAV939 is a small molecule inhibitor of tankyrase 1(TNKS1). The objective of this study is to investigate the effect of XAV939 on the proliferation and apoptosis of NB cell lines, and the related mechanism.
In the present study, we used both XAV939 treatment and RNAi method to demonstrate that TNKS1 inhibition may be a potential mechanism to cure NB. MTT method was used for determining the cell viability and the appropriate concerntration for follow-up assays. The colony formation assay, Annexin V staining and cell cycle analysis were used for detecting colony forming ability, cell apoptosis and the percentage of different cell cycle. The Western blot was used for detecting the expression of key proteins of Wnt/ beta-catenin (Wnt/β-catenin) signaling pathway.
The results showed that TNKS1 inhibition decreased the viability of SH-SY5Y, SK-N-SH and IMR-32 cells, induced apoptosis in SH-SY5Y as well as SK-N-SH cells, and led to the accumulation of NB cells in the S and G2/M phase of the cell cycle. Moreover, we demonstrated TNKS1 inhibition may in part blocked Wnt/β-catenin signaling and reduced the expression of anti-apoptosis protein. Finally, we also demonstrated that TNKS1 inhibition decreased colony formation in vitro.
These findings suggested that TNKS1 may be a potential molecule target for the treatment of NB.
Neuroblastoma; Tankyrase 1; Wnt signaling; XAV939
Paclitaxel, a widely used chemotherapeutic drug, can induce apoptosis in variety of cancer cells. A previous study has shown preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cell line, UOK257. In this report, we investigate the cellular and molecular mechanism of paclitaxel-induced autophagy and apoptosis in renal cancer cells with and without FLCN expression.
Two pairs of cell lines were used: FLCN siRNA-silenced ACHN cell line (ACHN-5968) and scrambled ACHN cell line (ACHN-sc); FLCN-null UOK257 cell line and UOK257-2 cell line restored with ectopic expression of FLCN. Autophagy was examined by western blot, GFP-LC3, transmission electron microscopy, and MDC assay. Cell viability and apoptosis were detected using MTT assay, DAPI stain and TUNEL assay. After inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, cell viability and apoptosis were measured by MTT assay and TUNEL assay.
After paclitaxel treatment, a dose-dependent decrease in cell viability and increase in apoptosis were observed in FLCN-deficient UOK257 and ACHN-5968 cells compared to their FLCN-expressing counterparts, suggesting that renal cancer cells without FLCN were more sensitive to paclitaxel. Enhanced autophagy was found to be associated with paclitaxel treatment in FLCN-deficient RCC cells. The MAPK pathway was also identified as a key pathway for the activation of autophagy in these kidney cancer cells. Inhibition of phosphorylated ERK with ERK inhibitor U0126 showed a significant decrease in autophagy. Furthermore, after inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, apoptosis induced by paclitaxel was significantly increased in FLCN-deficient UOK257 and ACHN-5968 cells.
Preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cells is associated with enhanced autophagy. Suppression of autophagy further enhances paclitaxel-induced apoptosis in FLCN-deficient renal cancer cells. Our results suggest that paclitaxel combined with an autophagy inhibitor might be a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer.
Autophagy; Apoptosis; Paclitaxel; Taxol; Folliculin; FLCN; BHD; Kidney cancer
Interleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells.
STAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors.
Our results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype.
IL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.
IL-27; STAT1; STAT3; Epithelial-mesenchymal transition; Cytokine; Angiogenesis
Similar to protein-coding genes, miRNAs are also susceptible to epigenetic modulation. Although numerous miRNAs have been shown to be affected by DNA methylation, the regulatory mechanism of histone modification on miRNA is not adequately understood. EZH2 and HDACs were recently identified as critical histone modifiers of deregulated miRNAs in cancer and can be recruited to a miRNA promoter by transcription factors such as MYC. Because miRNAs can modulate epigenetic architecture and can be regulated by epigenetic alteration, they could reasonably play an important role in mediating the crosstalk between epigenetic regulators. The complicated network between miRNAs and epigenetic machineries underlies the epigenetic–miRNA regulatory pathway, which is important in monitoring gene expression profiles. Regulation of miRNAs by inducing epigenetic changes reveals promising avenues for the design of innovative strategies in the fight against human cancer.
microRNA; Epigenetic; DNA methylation; Histone modification; Cancer
Fluoropyrimidines, oxaliplatin, irinotecan and biologic therapies (Bevacizumab, Panitumumab, and Cetuximab) represent the backbone of metastatic colorectal cancer (CRC) treatment. The improvement in survival for mCRC patient led to two main outstanding issues: 1) there is a significant number of patients progressing beyond the third or fourth line of treatment still suitable for further therapy when enrollment into clinical trial is not possible. In this situation, the role of any therapy rechallenge (either chemotherapy alone, chemotherapy and biologic therapy or biologic therapy alone) is still not clear, particularly in patients who had previously responded, and if treatment choice is based on traditional dogma of primary and secondary resistance, rechallenge does not seem to be justified. 2) Prolonged intensive treatment is burdened from the high risk of cumulative toxicity, worsening in quality of life and a not well defined possibility of early acquired resistance.
Different hypothesis could justify the research of different strategy in treatment of mCRC:
1) Epigenetic changes might drive resistance and treatment could induce these changes. Re-expression of silenced tumor suppressive genes might resensitize tumors to therapy. It is therefore possible that a drug holiday (intermittent treatment) could allow reversion to a previous epigenetic profile. Moreover an intermittent treatment could delay acquired resistance. 2) It is plausible that tumor grows as a polyclonal mass. If it responds but then becomes resistant to one or more treatments, retreatment might be successful if changing therapies allows to that clone of cells to re-emerge. On these basis, we focused this review on the actual evidences in management of mCRC patients in terms of chemotherapy or biological therapies rechallenge and intermittent treatment. Moreover, we will discuss the potential biological mechanisms of the observed results of early clinical trials.
Colorectal cancer; Drug resistance; Rechallenge therapy; Intermittent treatment
One of the key oncogenic pathways involved in melanoma aggressiveness, development and progression is the RAS/BRAF/MEK pathway, whose alterations are found in most patients. These molecular anomalies are promising targets for more effective anti-cancer therapies. Some Mek inhibitors showed promising antitumor activity, although schedules and doses associated with low systemic toxicity need to be defined. In addition, it is now accepted that cancers can arise from and be maintained by the cancer stem cells (CSC) or tumor-initiating cells (TIC), commonly expanded in vitro as tumorspheres from several solid tumors, including melanoma (melanospheres). Here, we investigated the potential targeting of MEK pathway by exploiting highly reliable in vitro and in vivo pre-clinical models of melanomas based on melanospheres, as melanoma initiating cells (MIC) surrogates. MEK inhibition, through PD0325901, provided a successful strategy to affect survival of mutated-BRAF melanospheres and growth of wild type-BRAF melanospheres. A marked citotoxicity was observed in differentated melanoma cells regardless BRAF mutational status. PD0325901 treatment, dramatically inhibited growth of melanosphere-generated xenografts and determined impaired tumor vascularization of both mutated- and wild type-BRAF tumors, in the absence of mice toxicity. These results suggest that MEK inhibition might represent a valid treatment option for patients with both mutated- or wild type-BRAF melanomas, affecting tumor growth through multiple targets.
Metastatic Melanoma; Mek inhibition; Melanospheres; Target therapy
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