Time-course expression of DNA repair-related genes in hepatocytes of zebrafish (Danio rerio) after UV-B exposure

The objective of this study was to evaluate the time-course effects of UV-B exposure on expression of genes involved in the DNA repair system of zebrafish ( Danio rerio ) hepatocytes, a highly competent species in terms of damage repair induced by UV radiation. For gene expression analysis (RT-PCR), cells were exposed to 23.3 mJ cm⁻² UV-B, which was the dose that affected viable cell number (reduction of 30% when compared with the control group) and produced no visual alteration on cell morphology. The early response observed (6 h) showed induction in the expression of the CDKI gene (cyclin-dependent kinase inhibitor) and genes related to DNA damage repair (mainly XPC and DDB2 ), while the late response observed (24 h) was more related to up-regulation of p53 and genes involved in cell cycle arrest ( gadd45a , cyclinG1 ). In all times analyzed, the anti-apoptotic gene Bcl-2 was down-regulated. Another interesting result observed was the up-regulation of the Apex- 1 gene after UV-B exposure, which could indicate the induction of oxidative lesions in the DNA molecule. In conclusion, these results demonstrate an activation of the DNA repair system in hepatocytes of zebrafish exposed to UV-B radiation, mainly involving the participation of p53.     

Expression of NDRG2 is related to tumor progression and survival of gastric cancer patients through Fas-mediated cell death

Although N-myc downstream regulated gene 2 (NDRG2) has been known to be a tumor suppressor gene, the function of this gene has not been elucidated. In the present study, we investigated the expression and function of NDRG2 in human gastric cancer. Among seven gastric cancer and two non-cancer cell lines, only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2, which was detected in the cytoplasm. Interestingly, NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and -negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. We confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis. To investigate the role of NDRG2 in gastric cancer cells, we generated a NDRG2-silenced gastric cancer cell line, which stably expresses NDRG2 siRNA. NDRG2-silenced SNU-620 cells exhibited slightly increased proliferation and cisplatin resistance. In addition, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. Taken together, these data suggest that inactivation of NDRG2 may elicit resistance against anticancer drug and Fas-mediated cell death. Furthermore, case studies of gastric cancer patients indicate that NDRG2 expression may be involved in tumor progression and overall survival of the patients.     

The function of p27KIP1 during tumor development

Timely cell cycle regulation is conducted by sequential activation of a family of serine-threonine kinases called cycle dependent kinases (CDKs). Tight CDK regulation involves cyclin dependent kinase inhibitors (CKIs) which ensure the correct timing of CDK activation in different phases of the cell cycle. One CKI of importance is p27^KIP1. The regulation and cellular localization of p27^KIP1 can result in biologically contradicting roles when found in the nucleus or cytoplasm of both normal and tumor cells. The p27^KIP1 protein is mainly regulated by proteasomal degradation and its downregulation is often correlated with poor prognosis in several types of human cancers. The protein can also be functionally inactivated by cytoplasmic localization or by phosphorylation. The p27^KIP1 protein is an unconventional tumor suppressor because mutation of its gene is extremely rare in tumors, implying the normal function of the protein is deranged during tumor development. While the tumor suppressor function is mediated by p27^KIP1''s inhibitory interactions with the cyclin/CDK complexes, its oncogenic function is cyclin/CDK independent, and in many cases correlates with cytoplasmic localization. Here we review the basic features and novel aspects of the p27^KIP1 protein, which displays genetically separable tumor suppressing and oncogenic functions.     

The Selectivity for Tumor Cells of Nuclear-Directed Cytotoxic RNases Is Mediated by the Nuclear/Cytoplasmic Distribution of p27KIP1

Although single targeted anti-cancer drugs are envisaged as safer treatments because they do not affect normal cells, cancer is a very complex disease to be eradicated with a single targeted drug. Alternatively, multi-targeted drugs may be more effective and the tumor cells may be less prone to develop drug resistance although these drugs may be less specific for cancer cells. We have previously developed a new strategy to endow human pancreatic ribonuclease with antitumor action by introducing in its sequence a non-classical nuclear localization signal. These engineered proteins cleave multiple species of nuclear RNA promoting apoptosis of tumor cells. Interestingly, these enzymes, on ovarian cancer cells, affect the expression of multiple genes implicated in metabolic and signaling pathways that are critic for the development of cancer. Since most of these targeted pathways are not highly relevant for non-proliferating cells, we envisioned the possibility that nuclear directed-ribonucleases were specific for tumor cells. Here, we show that these enzymes are much more cytotoxic for tumor cells in vitro. Although the mechanism of selectivity of NLSPE5 is not fully understood, herein we show that p27^KIP1 displays an important role on the higher resistance of non-tumor cells to these ribonucleases.     

Induction of G2/M Cell Cycle Arrest via p38/p21Waf1/Cip1-Dependent Signaling Pathway Activation by Bavachinin in Non-Small-Cell Lung Cancer Cells

Lung cancer is the most commonly diagnosed malignant cancer in the world. Non-small-cell lung cancer (NSCLC) is the major category of lung cancer. Although effective therapies have been administered, for improving the NSCLC patient’s survival, the incident rate is still high. Therefore, searching for a good strategy for preventing NSCLC is urgent. Traditional Chinese medicine (TCM) are brilliant materials for cancer chemoprevention, because of their high biological safety and low cost. Bavachinin, which is an active flavanone of Proralea corylifolia L., possesses anti-inflammation, anti-angiogenesis, and anti-cancer activities. The present study’s aim was to evaluate the anti-cancer activity of bavachinin on NSCLC, and its regulating molecular mechanisms. The results exhibited that a dose-dependent decrease in the cell viability and colony formation capacity of three NSCLC cell lines, by bavachinin, were through G2/M cell cycle arrest induction. Meanwhile, the expression of the G2/M cell cycle regulators, such as cyclin B, p-cdc2^Y15, p-cdc2^T161, and p-wee1, was suppressed. With the dramatic up-regulation of the cyclin-dependent kinase inhibitor, p21^Waf1/Cip1, the expression and association of p21^Waf1/Cip1 with the cyclin B/cdc2 complex was observed. Silencing the p21^Waf1/Cip1 expression significantly rescued bavachinin-induced G2/M cell accumulation. Furthermore, the expression of p21^Waf1/Cip1 mRNA was up-regulated in bavachinin-treated NSCLC cells. In addition, MAPK and AKT signaling were activated in bavachinin-added NSCLC cells. Interestingly, bavachinin-induced p21^Waf1/Cip1 expression was repressed after restraint p38 MAPK activation. The inhibition of p38 MAPK activation reversed bavachinin-induced p21^Waf1/Cip1 mRNA expression and G2/M cell cycle arrest. Collectively, bavachinin-induced G2/M cell cycle arrest was through the p38 MAPK-mediated p21^Waf1/Cip1-dependent signaling pathway in the NSCLC cells.     

Inactivation patterns of p16/INK4A in oral squamous cell carcinomas

The p16/INK4A is one of the major target genes in carcinogenesis and its inactivation has frequently been reported in other types of tumors. The purpose of this study is to evaluate inactivation patterns of p16/INK4A in oral squamous cell carcinoma. Six different oral cancer cell lines, SCC-4, SCC-9, SCC-15, SCC-25, KB, and SNUDH- 379 were examined for inactivation of p16/INK4A genes. In the analysis of p16/INK4A gene inactivation, PCR amplification, direct sequencing, and methylation-specific PCR methods were adopted for evaluation of homozygous deletion, point mutation, and promoter hypermethylation, respectively. Homozygous deletion was detected in SCC-25 and SCC-9. SCC-15 showed hypermethylated promoter region within p16/INK4A gene. It is suggestive in the present study that inactivation patterns of p16/INK4A were mainly homozygous deletion, promoter methylation rather than point mutation in oral squamous cancer cell lines, so treatment modalities of oral squamous cell carcinoma should be focused on these types of inactivation.     

Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2

For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.     

Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

分子信标用于p53 mRNA的体外定量检测

根据p53基因的序列设计并合成了能特异性检测p53 mRNA的分子信标(MB), 发展了一种快速定量测定细胞内总RNA提取物中p53 mRNA的方法. 采用鼻咽癌(CNE2)细胞系和经RNA干扰技术降低p53基因表达的CNE2-p53RNAi细胞系, 抽提总RNA并用MB检测, 验证了MB的检测对象是p53 mRNA. 将该方法应用于多种肿瘤细胞内p53基因表达水平的分析, 表达变化趋势与经典的mRNA分析方法RT-PCR检测结果相符. 在此基础上, 用MB对5-氟尿嘧啶(5-Fu)处理的肺腺癌细胞(A549)进行了p53 mRNA的体外定量检测, 结果表明采用MB能够快速地获知该药物对细胞内p53 mRNA表达影响的信息.     

A rapid fluorescence based multiplex polymerase chain reaction--single-strand conformation polymorphism method for p53 mutation detection

We have developed a fast single-strand conformation polymorphism (SSCP) technique to screen for mutations and polymorphisms in exons 5-8 of the human tumor suppressor gene p53. We use multiplex polymerase chain reaction (PCR) to amplify the four exons in one single PCR reaction and then fluorescent SSCP for screening. p53 fragments are labeled with three different colors and a fourth color is used for an internal size marker calibrating the gel. The method was evaluated in two ways: (i) 16 different cell lines with known mutations were tested blindly for band-shifts with SSCP, and (ii) 32 human urinary bladder cancer samples were screened for mutations using the present technique. After screening for mutations all exons from all samples were sequenced, both sense as well as antisense strands. Evaluating the method with four different gels shows that 21/23 mutations and polymorphism were detected in the cell lines and that 10/10 mutations and polymorphisms were detected in the patient samples. Sensitivity, specificity, positive and negative predictive values were 91/100%, 88/ 97%. 78/77% and 96/100% for cell lines / patient samples, respectively. Sensitivity, using one SSCP gel only, was 87% (20/23) for cell lines and 90% (9/10) for patient samples. We conclude that our modified SSCP technique is efficient and has a sensitivity close to 100% in detecting mutations.     

p53 overexpression represses androgen-mediated induction of NKX3.1 in a prostate cancer cell line

Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.     

DERL3 functions as a tumor suppressor in gastric cancer

BackgroundGastric cancer is a common malignant tumor in the clinic with a high mortality rate, ranking the first among malignant tumors of the digestive system. Early gastric cancer exhibits no specific clinical symptoms and signs, and most of the patients were diagnosed as advanced gastric cancer. The prognosis is poor, and the 5-year overall survival rate is still lower than 30%, seriously threatening people’s life and health. However, the pathogenesis of gastric cancer is still unclear.MethodsThis study aimed to identify methylated differentially expressed genes in gastric cancer and to study the cellular functions and pathways that may be involved in its regulation, as well as the biological functions of key methylated differentially expressed genes. The gene expression data set and methylation data set of gastric cancer genes based on TCGA were analyzed to identify prognostic methylated genes.ResultsThis study showed that the methylation of the DERL3 promoter was correlated with the clinical analysis of tumors. Further studies were conducted on genes co-expressed with DERL3, whose functions and pathways to inhibit gastric cancer were adaptive immune response, T cell activation, immune response-regulating pathway, cell surface on molecules, and natural killer cell-mediated cytotoxicity. Finally, cell proliferation assay, cell scratch assay, and cell invasion assay confirmed that DERL3 as a tumor suppressor gene inhibited the malignant evolution of gastric cancer.ConclusionsThe analysis of key methylated differentially expressed genes helped elucidate the epigenetic regulation mechanism in the development of gastric cancer. DERL3, as a methylation biomarker, has a predictive and prognostic value in the accurate diagnosis and treatment of gastric cancer and provides potential targets for the precision treatment of gastric cancer.Trial RegistrationNot applicable.     

Zinc oxide nanoparticles promotes ferroptosis to repress cancer cell survival and inhibits invasion and migration by targeting miR-27a-3p/YAP axis in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is a prevalent malignancy with growing mortality and high metastasis. Ferroptosis has been identified as an essential process in cancer development, but the regulatory mechanism underlying the RCC progression remains obscure. The nanomaterial zinc oxide nanoparticles (ZONs) have presented anti-cancer function. Here, we identified the critical role of ZONs in promoting ferroptosis of RCC cells by regulating miR-27a-3p/YAP axis.MethodsThe effect of ZONs on RCC was analyzed by qPCR, Western blot, MTT assays, colony formation assays, Flow cytometry analysis, transwell assays, wound healing assays, iron assays, lipid ROS detection, luciferase reporter gene assays, and tumor xenograft.ResultsThe treatment of ZONs repressed expression of GPX4 and SLC7A11 and enhanced ROS accumulation and iron/Fe²⁺ levels in RCC cells. Ferroptosis activator erastin repressed RCC cell viabilities and ZONs further repressed this effect. ZONs inhibited invasion and migration of RCC cells and treatment of ZONs represses RCC cell survival in vitro. ZONs suppressed RCC cell growth in tumorigenicity mouse model. Mechanically, ZONs down-regulated YAP expression by inducing miR-27a-3p, in which YAP overexpression and miR-27a-3p inhibition reverse ZONs -inhibited RCC cell survival in vitro.DiscussionThus, we concluded that ZONs induced RCC cell ferroptosis to suppress RCC cell survival by targeting miR-27a-3p/YAP axis. The clinical significance of ZONs for the treatment of RCC is required to further study and may benefit the targeted therapy of RCC.     

Low-dose radiation response of the p21WAF1/CIP1 gene promoter transduced by adeno-associated virus vector

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (< 1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.     

Acute response of human skin to solar radiation: regulation and function of the p53 protein.

p53 is a tumor suppressor gene and mutation of p53 is a frequent event in skin cancer. The wild-type p53 encodes for a 53-kD phosphoprotein that plays a pivotal role in regulating cell growth and cell death. The wt-p53 gene is also called "guardian of the genome", for its role in preventing the accumulation of genetic alterations, observed in cancer cells. The wild-type p53 protein plays a central role in the response of the cell to DNA damage. UV, present in sunlight, is one of the most ubiquitously present DNA damage inducing stress conditions to which skin cells are exposed. The wt-p53 protein accumulates in human skin cells in vitro and in human skin in vivo upon UV irradiation. This upregulation mounts a protective response against permanent DNA damage through transactivation of either cell cycle arrest genes and DNA repair genes or genes that mediate the apoptotic response. The molecular events which regulate the activity of the wt-p53 protein activity are only beginning to be described.