Suicide gene therapy is based on the introduction of a foreign gene into tumor cells to sensitize cells to treatment, to convert a nontoxic compound into a lethal drug, or to produce a cytotoxic effect. We have constructed a suicide gene therapy vector that contains resveratrol-responsive CArG elements from the Egr-1 promoter and the GADD45α open reading frame. CArG elements are utilized as a "molecular switch" to drive the expression of GADD45α. When transfected into lung cancer cells, the vector is able to express GADD45α upon resveratrol treatment, and subsequently leads to cell cycle arrest at the G2/M transition. In this chapter, we describe a detailed protocol for vector construction, transfection, cell viability assay, and cell cycle analysis.

The correlation between cigarette smoking and the onset of non-small cell lung cancer is well documented. Enhanced proliferation, angiogenesis induction, and resistance to apoptosis were reported as direct results associated with exposure to nicotine (the active ingredient of cigarettes). Parthenolide is a sesquiterpene lactone with anticancer activity against different cancer types. In this study, we tested the ability of parthenolide to inhibit the proliferating effect of nicotine in lung cancer cell lines. MTT assay was used to measure cell survival of A549 and H526 cells treated with nicotine, parthenolide, and their combination. Angiogenesis inhibition was measured using VEGF detection kit and apoptosis induction was evaluated by measuring caspase-3 activity. Real time PCR assay was used to detect the change in expression of several genes associated with cell proliferation and apoptosis (CASP3, CASP7, CASP8, CASP9, P53, GADD45, BAX, BIM, Bcl-2, TOPO I, and TOPO II). Parthenolide inhibited lung cancer cells in a concentration-dependent manner and decreased the proliferation stimulating effect of nicotine. Caspase-3 activity and VEGF assays evidenced an apoptosis-inducing and VEGF- inhibiting effects of parthenolide. The real time PCR assay demonstrated that parthenolide down-regulated the expression of Bcl-2 and up-regulated the expression of E2F1, P53, GADD45, BAX, BIM, and CASP 3,7,8,9, which indicates an activation of P53- dependent apoptosis pathway in response to parthenolide. Furthermore, this pathway remained active in the presence of nicotine suggesting the ability of parthenolide to exclude the anti-apoptotic effect of nicotine. Our results indicate that parthenolide inhibits nicotine proliferating effect on lung cancer. The anticancer effect of parthenolide is mediated by angiogenesis inhibition and activation of P53- dependent apoptosis. Parthenolide is a promising natural product for inhibiting and treating nicotine-associated lung cancer. However, further studied on more lung cancer cell lines and on protein level are needed to fully understand its mechanisms of action.

The efficacy of the anticancer drug cisplatin is restricted by tumor cell resistance and occurrence of severe side effects. One strategy to overcome these limitations is the development of new, improved platinum drugs. Previous investigations showed that platinum(IV)-nitroxyl complexes are able to circumvent cisplatin resistance in bladder cancer cells. In the present study the mode of action of the platinum(IV)-nitroxyl complex PN149 was investigated in the bladder cancer cell line RT112 and the renal cell carcinoma cell line A498 on the molecular and cellular level. Gene expression analysis showed that PN149 induced genes related to DNA damage response (RRM2B, GADD45A), cell cycle regulation (CDKN1A, PLK3, PPM1D) as well as those coding for the pro-apoptotic factors PUMA and Noxa. These findings on the transcriptional level were confirmed on the functional level revealing that PN149 treatment increased levels of p53 and resulted in cell cycle arrest and drug-induced cytotoxicity via induction of apoptosis. Regarding the expression of oxidative-stress sensitive genes, PN149 induced FTH1, GCLC, HMOX1 and TXNRD1 but relevant effects were restricted to RT112 cells treated with 50 µM. The pro-inflammatory IL-8 was induced by PN149 in RT112 but not A498 cells indicating a cell-type specific activation. Taken together, PN149 possessed promising activity in different tumor cell lines rendering it an interesting alternative to cisplatin in chemotherapy.

BACKGROUND: TP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102-292) and mainly reside in six "hotspot" residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53-/-.
RESULTS: MTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.
CONCLUSIONS: These results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.

BACKGROUND/AIMS: Triple-negative breast cancer (TNBC) is a highly aggressive malignancy that responds in a diverse manner to neoadjuvant chemotherapy (NAC). This study was aimed to uncover an RNA signature in TNBC patients which predicts pathological complete responses (pCR) to NAC by analyzing long noncoding RNA (lncRNA) and coding gene expression.
METHODS: Microarray datasets from 26 TNBC patients receiving NAC including ten patients showing pCR were obtained from the Gene Expression Omnibus database.
RESULTS: A total of 172 coding genes and 84 lncRNAs were differentially expressed between patients achieving pCR and those who did not. Filtering based on the predictive efficacy of response to NAC using receiver operator characteristic curve (ROC) and area under the curve (AUC) shortlisted 23 lncRNAs and 15 coding genes from consideration. Finally, a response score consisting of 1 lncRNA and 2 coding genes was developed: response score = 2.595*BPESC1 - 1.09*WDR72 -1.428*GADD45A - 0.731. The response score had good predictive performance (AUC=0.931, p< 0.01) and at the cut-off of 0.545, the response score had sensitivity and specificity of 0.8 and 0.9, respectively.
CONCLUSION: We propose a simple gene expression signature of only three RNA species could be employed clinically to predict pCR in TNBC patients receiving NAC.

BACKGROUND: Glioblastoma (GBM) is the most common, malignant, and lethal primary brain tumor in adults accounting for about 50% of all gliomas. Up to now, the chemotherapy approaches for GBM were limited. 3-O-acetyl-11-keto-β-boswellic acid (AKBA), the major active ingredient of the gum resin from Boswellia serrata and Boswellia carteri Birdw., was reported to inhibit the growth of many types of cancer cells; however, the underlying mechanism of its anticancer effects are still unclear.
METHODS: The effects of AKBA on cell viability and its cytotoxicity were determined using CCK8 and LDH kits respectively. The EdU-DNA synthesis assay was used to evaluate inhibition of cell proliferation by AKBA. The role of AKBA in glioblastoma cell functions such as migration/invasion, and colony formation was evaluated using transwell chambers and soft agar, respectively. Flow cytometry and western blotting were used to detect AKBA-induced apoptosis. Potential mechanisms of AKBA action were explored by RNA sequencing and the identified hub genes were validated by real-time quantitative PCR and western blotting. Finally, the in vivo anti-tumor activity of AKBA was evaluated against a human glioblastoma cell line, U87-MG, in a xenograft mouse model.
RESULTS: AKBA inhibited cell proliferation, caused the release of LDH, decreased DNA synthesis, and inhibited the migration, invasion, and colony formation of U251 and U87-MG human glioblastoma cell lines. AKBA increased apoptosis as well as the activity of caspase 3/7 and the protein expression of cleaved-caspase 3 and cleaved PARP, while decreasing mitochondrial membrane potential. RNA-sequencing analyses showed that AKBA suppressed the expression of pRB, FOXM1, Aurora A, PLK1, CDC25C, p-CDK1, cyclinB1, Aurora B, and TOP2A while increasing the expression of p21 and GADD45A. These findings were validated by qRT-PCR and western blotting. The data are consistent with a mechanism in which AKBA arrested the cell cycle in glioblastoma cells at the G2/M phase by regulating the p21/FOXM1/cyclin B1 pathway, inhibited mitosis by downregulating the Aurora B/TOP2A pathway, and induced mitochondrial-dependent apoptosis. Oral administration of AKBA (100 mg/kg) significantly suppressed the tumorigenicity of U87-MG cells in a xenograft mouse model.
CONCLUSIONS: Taken together, these results suggest that AKBA (molecular weight, 512.7 Da) might be a promising chemotherapy drug in the treatment of GBM.

Curcumin is a potential anticancer drug with poor bioavailability, which limits its clinical use as a therapeutic agent. The aim of this study was a preliminary evaluation of the curcumin analogue CH-5 as a cytotoxic agent in human osteosarcoma cell lines U2OS, MG-63, and Saos-2. CH-5 inhibited cell viability at lower concentrations than curcumin, leading to the induction of apoptosis. The cellular levels of the transcription factors p53 and Sp1 affect the expression of cellular pathways that lead to apoptosis. CH-5 increased p53 protein levels in U2OS cells and reduced Sp1 levels, with a consequent effect on the expression of their target genes DNA methyltransferase 1 (

Nickel is a major carcinogen that is implicated in tumor development through occupational and environmental exposure. Although the exact molecular mechanisms of carcinogenesis by low-level nickel remain unclear, inhibition of DNA repair is frequently considered to be a critical mechanism of carcinogenesis. Here, we investigated whether low concentrations of nickel would affect p53-mediated DNA repair, especially nucleotide excision repair. Our results showed that nickel inhibited the promoter binding activity of p53 on the downstream gene GADD45A, as a result of the disturbance of p53 oligomerization by nickel. In addition, we demonstrated that nickel exposure trigger the reduction of GADD45A-mediated DNA repair by impairing the physical interactions between GADD45A and proliferating cell nuclear antigen or xeroderma pigmentosum G. Notably, in the GADD45A-knockdown system, the levels of unrepaired DNA photoproducts were higher than wild-type cells, elucidating the importance of GADD45A in the nickel-associated inhibition of DNA repair. These results imply that inhibition of p53-mediated DNA repair can be considered a potential carcinogenic mechanism of nickel at low concentrations.

There is limited data on the molecular mechanisms whereby hepatitis D virus (HDV) promotes liver cancer. Therefore, serum and liver specimens obtained at the time of liver transplantation from well-characterized patients with HDV-HCC (

Transcriptional dosimetry is an emergent field of radiobiology aimed at developing robust methods for detecting and quantifying absorbed doses using radiation-induced fluctuations in gene expression. A combination of RNA sequencing, array-based and quantitative PCR transcriptomics in cellular, murine and various ex vivo human models has led to a comprehensive description of a fundamental set of genes with demonstrable dosimetric qualities. However, these are yet to be validated in human tissue due to the scarcity of in situ-irradiated source material. This represents a major hurdle to the continued development of transcriptional dosimetry. In this study, we present a novel evaluation of a previously reported set of dosimetric genes in human tissue exposed to a large therapeutic dose of radiation. To do this, we evaluated the quantitative changes of a set of dosimetric transcripts consisting of FDXR, BAX, BCL2, CDKN1A, DDB2, BBC3, GADD45A, GDF15, MDM2, SERPINE1, TNFRSF10B, PLK3, SESN2 and VWCE in guided pre- and post-radiation (2 weeks) prostate cancer biopsies from seven patients. We confirmed the prolonged dose-responsivity of most of these transcripts in in situ-irradiated tissue. BCL2, GDF15, and to some extent TNFRSF10B, were markedly unreliable single markers of radiation exposure. Nevertheless, as a full set, these genes reliably segregated non-irradiated and irradiated tissues and predicted radiation absorption on a patient-specific basis. We also confirmed changes in the translated protein product for a small subset of these dosimeters. This study provides the first confirmatory evidence of an existing dosimetric gene set in less-accessible tissues-ensuring peripheral responses reflect tissue-specific effects. Further work will be required to determine if these changes are conserved in different tissue types, post-radiation times and doses.

Baicalein has efficient antitumor properties and has been reported to promote the apoptosis of several human cancer cell lines. Decidual protein induced by progesterone (DEPP), a transcriptional target of Forkhead Box O, was originally identified from the human endometrial stromal cell cDNA library. However, the expression and physiological functions of DEPP in human colon cancer cells remain to be fully elucidated. In the present study, it was reported that baicalein stimulated apoptosis and morphological changes of HCT116, A549 and Panc‑1 cells in a dose-dependent manner. It also upregulated the mRNA and protein levels of DEPP and growth arrest and DNA damage-inducible 45α (Gadd45a). In addition, the overexpression of DEPP promoted mitogen-activated protein kinase (MAPK) phosphorylation. To further investigate the role of DEPP and Gadd45a in baicalein-induced apoptosis, HCT116 cells were transfected with small interfering RNA against either DEPP or Gadd45a as in vitro models. Through an Annexin V/PI double staining assay, it was observed that baicalein-induced apoptosis was impaired by the inactivation of either DEPP or Gadd45a, which in turn restricted the baicalein-induced activation of caspase‑3 and caspase‑9 and phosphorylation of MAPKs. In addition, the inhibition of c‑Jun N‑terminal kinase (JNK)/p38 activity with SP600125/SB203580 decreased the expression of Gadd45a, whereas the inactivation of extracellular signal-regulated kinase with SCH772984 had no effect on the expression of Gadd45a. Taken together, these results demonstrated that baicalein induced the upregulation of DEPP and Gadd45a, which promoted the activation of MAPKs with a positive feedback loop between Gadd45a and JNK/p38, resulting in a marked apoptotic response in human colon cancer cells. These results indicated that baicalein is a potential antitumor drug for the treatment of colon cancer.

Exposure to pesticides results in DNA damage and genomic instability. We previously predicted that endosulfan might be associated with leukemia, but the role of endosulfan in leukemia cells has been unexplored. The aim of this study is to elucidate molecular mechanism of endosulfan-induced DNA damage response in human leukemia cells. We performed endosulfan exposure experiments in K562 cells with varying concentrations of endosulfan for 48 h and found that endosulfan lowered cell viability in a dose-dependent manner. We observed the dramatic DNA damage using comet assay and the increase of micronucleus in 75 μM endosulfan-exposed cells. Endosulfan at 75 μM caused the expression alterations of ATM and DNA repair genes such as FANCD2, and BRCA1/2 at different exposure time points (12, 24, 48 h), which was reversed by ATM inhibitor KU-55933. Endosulfan significantly increased the mRNA expression levels of p53 and GADD45A, and decreased PCNA and XRCC2 at 48 h after exposure. Flow cytometric analysis showed that endosulfan at 50 and 75 μM induced cell cycle G1 arrest, a response attributed to down-regulation of CDK6 and up-regulation of p21. We also observed that endosulfan at 50 and 75 μM induced a considerable percentage of cells to undergo apoptosis, as detected by Annexin-V binding assays. Endosulfan resulted in the activation of caspase-3, and elevated the expression levels of PUMA and the ratio of BAX/Bcl-2. These findings suggest that endosulfan caused DNA damage response throughATM-p53 signaling pathway, implicating the potential correlation between endosulfan and leukemia.

The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing new, rapid and high throughput biodosimetric tools for estimation of received dose and initial triage. We aimed to compare the induction and persistence of different radiation exposure biomarkers in human peripheral blood in vivo. Blood samples of patients with indicated radiotherapy (RT) undergoing partial body irradiation (PBI) were obtained soon before the first treatment and then after 24 h, 48 h, and 5 weeks; i.e. after 1, 2, and 25 fractionated RT procedures. We collected circulating peripheral blood from ten patients with tumor of endometrium (1.8 Gy per fraction) and eight patients with tumor of head and neck (2.0-2.121 Gy per fraction). Incidence of dicentrics and micronuclei was monitored as well as determination of apoptosis and the transcription level of selected radiation-responsive genes. Since mitochondrial DNA (mtDNA) has been reported to be a potential indicator of radiation damage in vitro, we also assessed mtDNA content and deletions by novel multiplex quantitative PCR. Cytogenetic data confirmed linear dose-dependent increase in dicentrics (p < 0.01) and micronuclei (p < 0.001) in peripheral blood mononuclear cells after PBI. Significant up-regulations of five previously identified transcriptional biomarkers of radiation exposure (PHPT1, CCNG1, CDKN1A, GADD45, and SESN1) were also found (p < 0.01). No statistical change in mtDNA deletion levels was detected; however, our data indicate that the total mtDNA content decreased with increasing number of RT fractions. Interestingly, the number of micronuclei appears to correlate with late radiation toxicity (r2 = 0.9025) in endometrial patients suggesting the possibility of predicting the severity of RT-related toxicity by monitoring this parameter. Overall, these data represent, to our best knowledge, the first study providing a multiparametric comparison of radiation biomarkers in human blood in vivo, which have potential for improving biological dosimetry.

BACKGROUND: Prohibitin 1 (PHB) is a potential target for the treatment of urothelial carcinoma of the bladder (UCB). FL3 is a newly synthesized agent that inhibits cancer cell proliferation by targeting the PHB protein; however, the effect of FL3 in UCB cells remains unexplored.
METHODS: FL3 was identified to be a potent inhibitor of UCB cell viability using CCK-8 (cell counting kit-8) assay. Then a series of in vitro and in vivo experiments were conducted to further demonstrate the inhibitory effect of FL3 on UCB cell proliferation and to determine the underlying mechanisms.
RESULTS: FL3 inhibited UCB cell proliferation and growth both in vitro and in vivo. By targeting the PHB protein, FL3 inhibited the interaction of Akt and PHB as well as Akt-mediated PHB phosphorylation, which consequently decreases the localization of PHB in the mitochondria. In addition, FL3 treatment resulted in cell cycle arrest in the G2/M phase, and this inhibitory effect of FL3 could be mimicked by knockdown of PHB. Through the microarray analysis of mRNA expression after FL3 treatment and knockdown of PHB, we found that the mRNA expression of the growth arrest and DNA damage-inducible alpha (GADD45α) gene were significantly upregulated. When knocked down the expression of GADD45α, the inhibitory effect of FL3 on cell cycle was rescued, suggesting that FL3-induced cell cycle inhibition is GADD45α dependent.
CONCLUSION: Our data provide that FL3 inhibits the interaction of Akt and PHB, which in turn activates the GADD45α-dependent cell cycle inhibition in the G2/M phase.

DNA methylation is a significant epigenetic modification which plays a key role in regulation of gene expression and influences functional changes in endometrial tissue. Aberrant DNA methylation changes result in deregulation of important apoptotic proteins during endometrial carcinogenesis and apoptosis resistance development. Evading apoptosis is still a major problem in the successful treatment of endometrial cancer patients. The aim of our study was to examine the promoter DNA methylation changes in 22 apoptosis-associated genes in endometrioid endometrial cancer patients, precancerous lesions and healthy tissue from various normal menstrual cycle phases using a unique pre-designed methylation platform. We observed as the first a significant difference in promoter DNA methylation status in genes: BCL2L11 (p < 0.001), CIDEB (p < 0.03) and GADD45A (p < 0.05) during endometrial carcinogenesis and BIK gene (p < 0.03) in different phases of normal menstrual cycle. The results of our study indicate that deregulation of mitochondrial apoptotic pathway can considerably contributes to the apoptosis resistance development and may be helpful in identifying of new potent biomarkers in endometrial cancer.

BACKGROUND: Development of resistance to therapy continues to be a serious clinical problem in lung cancer management. We previously identified that Annexin A2 is significantly up-regulated in cisplatin-resistant non-small cell lung cancer (NSCLC) A549/DDP cells. However, the exact function and molecular mechanism of Annexin A2 in cisplatin resistance of NSCLCs has not been determined.
METHODS: Western blot and qRT-PCR were performed to analyze the protein and mRNA level of indicated molecules, respectively. Immunohistochemistry was performed to analyze the expression of Annexin A2 in NSCLC tissue samples. MTS assay, Colony formation assays, AnnexinV/PI apoptosis assay, Luciferase Reporter Assay, Chromatin-immunoprecipitation, and nude mice xenograft assay were used to visualize the function of Annexin A2 on cisplatin resistance.
RESULTS: Our results demonstrated that knockdown of Annexin A2 increased cisplatin sensitivity of cisplatin-resistant A549/DDP cells both in vitro and in vivo, whereas overexpression of Annexin A2 increased cisplatin resistance of A549, H460 and H1650 cells. Moreover, we found that Annexin A2 enhanced cisplatin resistance via inhibition of cisplatin-induced cell apoptosis. Our studies showed that Annexin A2 suppressed the expression of p53 through activation of JNK/c-Jun signaling, which in turn resulted in a decrease in the expression of p53-regulated apoptotic genes p21, GADD45 and BAX, as well as p53-dependent cell apoptosis. Furthermore, we found that in NSCLC cases that Annexin A2 is highly expressed; it is positively correlated with a poor prognosis, as well as correlated with short disease-free survival for patients who received chemotherapy after surgery.
CONCLUSIONS: These data suggested that Annexin A2 induces cisplatin resistance of NSCLCs via regulation of JNK/c-Jun/p53 signaling, and provided an evidence that blockade of Annexin A2 could serve as a novel therapeutic approach for overcoming drug resistance in NSCLCs.

Lung cancer is the most common cause of cancer-related mortality worldwide. The GADD45 gene family plays important roles in a variety of the responses to cell injury including cell cycle checkpoints, apoptosis, DNA repair and anti-tumor immunity. The 19p13.3-GADD45B encoded protein product is involved in apoptosis and inhibiting tumor growth. To evaluate the association of 19p13.3-GADD45B common variants and lung cancer risk, the present study containing 544 Chinese lung cancer cases and 550 cancer-free controls was conducted. Three htSNPs (haplotype-tagging single nucleotide polymorphism) (rs7354, rs14384, and rs3783501) covering 95% of the common haplotype diversity in 19p13.3-GADD45B and interaction of 19p13.3-GADD45B and 19q13.3-PPP1R13L and 19q13.3-CD3EAP variants and smoking-duration were explored. Genotype and allele frequencies and haplotype distributions of the 19p13.3-GADD45B 3 htSNPs were not associated with lung cancer risk after adjustment for smoking status. 19p13.3-GADD45B rs7354 was associated with lung cancer risk among ≤20 (years) smokers [C/A-A/A versus CC, OR (95% CI) = 3.20 (1.11-9.20), P = 0.025] in a dominant model stratified by smoking duration. MDR (multifactor dimensionality reduction) analyses showed that smoking history as main effect and three-way models (smoking duration, 19p13.3-GADD45B rs3783501, 19q13.3-CD3EAP rs967591) (P = 0.001-0.002) indicated statistically significant association with lung cancer risk. The study identified evidence implicating DNA damage response genes on chromosome 19 in etiology of smoke-exposed lung cancer. In conclusion, our findings demonstrate that 19p13.3-GADD45B rs7354 variant and interaction between 19p13.3-GADD45B rs3783501 and 19q13.3-CD3EAP rs967591 may play a role in association with smoke-exposed lung cancer among Chinese. 19p13.3-GADD45B variants should be further evaluated in large prospective studies with molecular pathological annotations of lung cancer.

Glioblastoma multiforme (GBM) is one of the most aggressive cancers. Despite recent advances in multimodal therapies, high-grade glioma remains fatal. Temozolomide (TMZ) is an alkylating agent used worldwide for the clinical treatment of GBM; however, the innate and acquired resistance of GBM limits its application. Here, we found that TMZ inhibited the proliferation and induced the G2/M arrest of GBM cells. Therefore, we performed microarrays to identify the cell cycle- and apoptosis-related genes affected by TMZ. Notably, GADD45A was found to be up-regulated by TMZ in both cell cycle and apoptosis arrays. Furthermore, GADD45A knockdown (GADD45A

Neurotoxicity is known to be a major dose-limiting adverse effect of cisplatin (CDDP), alone or in combination with other chemicals. DNA repair capacity serve as a neuroprotective factor against CDDP. The purpose of this study was to evaluate the effect of 50-Hz electromagnetic field (EMF) in combination with CDDP and bleomycin (Bleo) on expression of some of DNA repair genes (GADD45A, XRCC1, XRCC4, Ku70, Ku80, DNA-PKcs and LIG4) in MCF-7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. MCF-7 and SH-SY5Y cells were pre-treated with CDDP in the presence or absence of EMF and then exposed to different concentration of Bleo. EMF (0.50mT intensity) was used in the intermittenet pattern of "15min field on/15min field off" with 30min total exposure. Cell viability assay was done and then the transcript levels of the examined genes were measured using quantitative real-time PCR in "CDDP+Bleo" and "CDDP+EMF+Bleo" treatments. Our results indicated that MCF-7 cells treated with "CDDP+EMF+Bleo" showed more susceptibility compared with "CDDP+Bleo" treated ones, while SH-SY5Y susceptibility was not changed between the two treatments. The represented data indicated that MCF-7 and SH-SY5Y cells showed non-random disagreement in DNA repair gene expression in 11 conditions (out of 14 conditions) with each other (χ

GADD45A (growth arrest and DNA damage inducible alpha), a stress response gene induced by genotoxic and nongenotoxic stresses, is implicated in various key processes, including the control of cell cycle checkpoints and DNA repair. The expression of GADD45A is directly regulated by numerous transcription factors, with p53 being the most representative. Moreover, post-transcriptional regulation also plays a role in GADD45A expression. However, little is known about the regulatory effects of microRNAs (miRNAs) on GADD45A expression. As a potential tumour suppressor, miR-138 has pleiotropic biological functions in various cancers. We have previously reported p53-mediated activation of miR-138 in human non-small-cell lung cancer (NSCLC) cells. In this study, we found that miR-138 specifically targeted AGO2, which affects the stability and maturation of miR-130b. Decreased expression of miR-130b promoted the expression of GADD45A and resulted in the G2/M phase arrest and proliferation inhibition in human NSCLC cells. Our results suggested that p53 could alternatively upregulate GADD45A in human NSCLC cells through a post-transcriptional pathway in which miR-138 is involved.

Aberrant DNA methylation patterns are a common theme across all cancer types. Specific DNA demethylation of regulatory sequences can result in upregulation of genes that are critical for tumor development and progression. Integrin α6β4 is highly expressed in pancreatic carcinoma and contributes to cancer progression, in part, through the specific DNA demethylation and upregulation of epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG). Whole genome bisulfite sequencing (WGBS) revealed that integrin α6β4 signaling promotes an overall hypomethylated state and site specific DNA demethylation of enhancer elements within the proximal promoters of AREG and EREG. Additionally, we find that the base excision repair (BER) pathway is required to maintain expression of AREG and EREG, as blocking DNA repair molecules, TET1 GADD45A, TDG, or PARP-1 decreased gene expression. Likewise, we provide the novel finding that integrin α6β4 confers an enhanced ability on cells to repair DNA lesions and survive insult. Therefore, while many known signaling functions mediated by integrin α6β4 that promote invasive properties have been established, this study demonstrates that integrin α6β4 can dramatically impact the epigenome of cancer cells, direct global DNA methylation levels toward a hypomethylated state, and impact DNA repair and subsequent cell survival.

Aberrant regulation of BCL6 plays crucial oncogenic roles in various malignant tumors; howbeit, the function of BCL6 in tumorigenesis of ovarian cancer remains unclear. The aim of this study is to investigate the role of BCL6 in ovarian cancer. The methods of immunohistochemical staining, quantitative real-time polymerase chain reaction, immunocytochemical staining, and gene expression profile enrichment analysis were performed to identify the possible role of BCL6 in ovarian cancer. We observed that the expression of BCL6 was significantly higher in ovarian cancer tissues and correlated with higher tumor burden including advanced International Federation of Gynecology and Obstetrics stages, poor differentiation, Type II ovarian cancer, the presence of >1 cm residual tumor size, and appearance of recurrence or death (all p < 0.05). The expression patterns of Lewis y were similar to these of BCL6. Multivariate Cox analysis demonstrated that advanced International Federation of Gynecology and Obstetrics stage, lymph node metastasis, residual tumor size >1 cm, as well as high expressions of BCL6 and Lewis y antigen were independent factors of worse progression-free survival and overall survival (all p < 0.05). There was a positive correlation of the expressions of BCL6 and Lewis y antigen. The associated genes with BCL6 in response to Lewis y antigen were identified, including four upregulated genes ( SOCS3, STAT1, PPARG, and GADD45A) and three downregulated genes ( ACAN, E2F3, and ZBTB7B). In conclusion, the high expressions of BCL6 and Lewis y antigen are associated with development, high tumor burden, and worse prognosis of ovarian cancer and targeting BCL6 could be a novel therapeutic strategy for ovarian cancer treatment.

Simultaneous use of cisplatin (CIS) and gemcitabine (GEN) for treating bladder cancer has increased because of their complementary effects. However, the molecular mechanisms underlying the activities of these two antineoplastic drugs are not fully known. Here, molecular biology techniques and microscopy were used to investigate transcriptomic and morphological changes in low and high-grade urinary bladder transitional carcinoma cell lines [RT4 - wild type TP53; 5637 - two TP53 mutations, one in codon 72 (Arg-Pro) and other in codon 280 (Arg-Thr) and T24 - in-frame deletion of tyrosine 126 in the TP53 allele] simultaneously treated with CIS/GEN. Gene expression profile was evaluated by PCR arrays; cell morphology by scanning and transmission electron microscopy, and apoptosis was analyzed using fluorescent dye. Results showed concomitantly upregulation of CDKN2B (G1/S transition), GADD45A (DNA repair and apoptosis) and SERTAD1 (regulation of transcription) gene, increased number of nuclear chamfers and apoptotic cells, and reduced number of microfilaments, organelles and in the size of the nucleus in 5637 and T24 cells after simultaneous treatment with CIS/GEN. In conclusion, independently of the TP53 mutation status and tumor grade, CIS/GEN induced gene modulation accompanied by changes in cell morphologies, which confirm the antiproliferative activity of the treatment protocol. These findings help to understand the pathways modulated by these antineoplastic agents and may provide insights for anti-cancer chemotherapy.

Prostate cancer (PCa) is the second leading cause of death from cancer in men. The mechanism underlying tumorigenesis and development of PCa is largely unknown. Here, we identified Kinesin family member 14 (KIF14) as a novel candidate oncogene in PCa. We found that KIF14 was overexpressed in multiple PCa cell lines and primary PCa tissues. Knockdown of KIF14 in DU145 and PC3 prostate cancer cells suppressed cell proliferation, induced cell cycle arrest and apoptosis. Transcriptome analysis by RNA-sequencing demonstrated that KIF4 suppression led to transcriptional changes of genes involved in p53 and TGF-beta signaling pathway. In addition, upregulated expression of GADD45A, GADD45B, p21, PIDD and Shisa5, which contribute to growth arrest and apoptosis induction, and downregulated CCNB1 that promotes cell cycle progression were confirmed by quantitative real-time PCR after KIF4 knockdown. We further found that KIF14 protein level was positively correlated with T stage and Gleason Score. Patients with higher KIF14 expression had shorter overall survival time than those with lower KIF14 expression. Thus, our data indicate that KIF14 could act as a potential oncogene that contributes to tumor progression and poor prognosis in PCa, which may represent a novel and useful prognostic biomarker for PCa.

Nonmelanoma skin cancer (NMSC) is the most common malignancy in the United States representing a considerable public health burden. Pharmacological suppression of skin photocarcinogenesis has shown promise in preclinical and clinical studies, but more efficacious photochemopreventive agents are needed. Here, we tested feasibility of harnessing pharmacological disruption of intracellular zinc homeostasis for photochemoprevention in vitro and in vivo. Employing the zinc ionophore and FDA-approved microbicidal agent zinc pyrithione (ZnPT), used worldwide in over-the-counter (OTC) topical consumer products, we first demonstrated feasibility of achieving ZnPT-based intracellular Zn

High-grade gliomas are severe tumors with poor prognosis. An R132H mutation in the isocitrate dehydrogenase (IDH1) gene prolongs the life of glioma patients. In this study, we investigated which genes are differentially regulated in IDH1 wild type (IDH1WT) or IDH1 R132H mutation (IDH1R132H) glioblastoma cells. Growth arrest and DNA-damage-inducible protein (GADD45A) was downregulated and microRNA 148a (miR-148a) was upregulated in in IDH1R132H human glioblastomas tissues. The relationship between GADD45A and miR-148a is unknown. In vitro experiments showed that GADD45A negatively regulates IDH1R132H glioma cell proliferation, migration, and invasion, and neurosphere formation in IDH1R132H glioblastoma stem cells (GSC). In addition, a human orthotopic xenograft mouse model showed that GADD45A reduced tumorigenesis in vivo. Our findings demonstrated that miR-148a promotes glioma cell invasion and tumorigenesis by downregulating GADD45A. Our findings provide novel insights into how GADD45A is downregulated by miR-148a in IDH1R132H glioma and may help to identify therapeutic targets for the effective treatment of high-grade glioma.

A number of significant studies in the field of cell biology have revealed another pattern of intracellular signal transduction in which cells transmit information through the dynamics of key signaling molecules. Dynamical properties of p53 have been demonstrated to be the key factor in dictating cell fate, including cell cycle arrest, permanent cell cycle arrest, and cell death. Previous studies showed a negative feedback regulation pathway between SGK1 and p53, but the dynamics of SGK1 have never been reported before. Therefore, we used different dosing strategies of Wogonin to affect SGK1 dynamics and investigate its impact on cell response. Key factors, such as APAF1, BAX, GADD45A, p21, PML, and YPEL3, which are related to cell cycle arrest, senescence, and apoptosis, were measured at different time points after incubation with Wogonin. Western blot and quantitative reverse transcriptase-polymerase chain reaction analysis were used to examine protein and mRNA expression of these genes. In addition, we also used β-galactosidase staining and flow cytometric analysis to further verify the results. It was found that Wogonin inhibited cell viability and downregulated SGK1 protein levels; 20 μM Wogonin could induce non-small-cell lung cancer A549 cells into cell cycle arrest/senescence/apoptosis after 0.5/2/4 h, respectively; and SGK1 dynamics showed significant differences under different cell responses. Together, our findings showed that SGK1 protein dynamics can be an important part of intracellular signaling, directly influencing cellular response decisions.

Multiple myeloma (MM) is a common hematological malignancy. To identify key genes and microRNAs in MM, we downloaded two gene expression profiles (GSE16558 and GSE47552) and two microRNA expression profiles (GSE17498 and GSE16558) from the Gene Expression Omnibus (GEO) database. A total of 596 differentially expressed genes (DEGs) and 39 differentially expressed microRNAs (DEMs) were screened out. Pathway analysis showed that upregulated genes were mainly enriched in the "B cell receptor signaling pathway", "Cell cycle" and "NF-kappa B signaling pathway", whereas downregulated genes were mainly enriched in the "Ribosome", "FoxO signaling pathway" and "p53 signaling pathway". We subsequently constructed a protein-protein interaction network of DEGs consisting of 277 genes and 563 interactions. In addition, 32 genes with high degrees in the network were identified as hub genes in MM, e.g. HDAC2, RBBP4, CREB1, and RB1. Additionally, we constructed a microRNA-mRNA regulatory network depicting interactions between DEMs and their targets, including the miR-135b-GADD45A and miR-148a-USPL1 pairs. In conclusion, the results of this data mining and integration help reveal the molecular basis of MM pathogenesis as well as potential biomarkers and therapeutic targets for MM diagnosis and treatment.

The Gadd45a stress sensor gene is a member in the Gadd45 family of genes that includes Gadd45b & Gadd45g. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45a null myeloid progenitors transduced with a retroviral vector expressing the 210-kD BCR-ABL fusion oncoprotein. Loss of Gadd45a was observed to accelerate BCR-ABL driven CML resulting in the development of a more aggressive disease, a significantly shortened median mice survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin- cKit+Sca+). GADD45A deficient progenitors expressing BCR-ABL exhibited increased proliferation and decreased apoptosis relative to WT counterparts, which was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPα expression, and hyper-activation of p38 and Stat5. Furthermore, Gadd45a expression in samples obtained from CML patients was upregulated in more indolent chronic phase CML samples and down regulated in aggressive accelerated phase CML and blast crisis CML. These results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may provide a unique prognostic marker of CML progression.

Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes.