Enhanced induction of Bax gene expression in H460 and H1299 cells with the combined treatment of cisplatin and adenovirus mediated wt-p53 gene transfer

Cytotoxic effect of either cisplatin or p53 gene transfection of lung cancer cells may be different depending on the p53 status of cells. We investigated cytotoxic effects on the combined treatment of cisplatin and adenovirus mediated p53 gene transfer (Avp53) in both H460 and H1299 cells in vitro. The results showed the highest numbers of apoptotic cells in both H460 and H1299 cells following the combined treatment regardless of p53 status in comparison with either cisplatin or Avp53 alone. The expression levels of p53, p21, Bax and ICE were examined to understand a possible cellular signal path of the combined treatment. In western analyses, the patterns of phosphorylated p53 protein were different between Avp53 and combined treatment. The expressions of p21 and Bax were increased in combined treatment, whereas the cleaved form of ICE (20 kD) was not detected. These results suggest that cisplatin induced p53 protein phosphorylation and may activate the downstream of p53 gene expression such as p21 and Bax. The enhanced apoptosis of lung cancer cells by the combined treatment may be useful in the development of clinical therapeutic modality of lung tumors.     

Functional characterization of human oncoprotein gankyrin in Zebrafish

Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma (HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2-hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation.     

Involvement of mitogen-activated protein kinases and p21Waf1 in hydroxyurea-induced G1 arrest and senescence of McA-RH7777 rat hepatoma cell line

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21(Waf1), p27(Kip1) and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21(Waf1) was increased, while p27(Kip1) and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21(Waf1) over-expression.     

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.     

HDAC Inhibitory and Anti-Cancer Activities of Curcumin and Curcumin Derivative CU17 against Human Lung Cancer A549 Cells

Previous research reported that the curcumin derivative (CU17) inhibited several cancer cell growths in vitro. However, its anticancer potential against human lung cancer cells (A549 cell lines) has not yet been evaluated. The purpose of this research was to examine the HDAC inhibitory and anti-cancer activities of CU17 compared to curcumin (CU) in A549 cells. An in vitro study showed that CU17 had greater HDAC inhibitory activity than CU. CU17 inhibited HDAC activity in a dose dependent manner with the half-maximal inhibitory concentration (IC₅₀) value of 0.30 ± 0.086 µg/mL against HDAC enzymes from HeLa nuclear extract. In addition, CU17 could bind at the active pockets of both human class I HDACs (HDAC1, 2, 3, and 8) and class II HDACs (HDAC4, 6, and 7) demonstrated by molecular docking studies, and caused hyperacetylation of histone H3 (Ac-H3) in A549 cells shown by Western blot analysis. MTT assay indicated that both CU and CU17 suppressed A549 cell growth in a dose- and time-dependent manner. Besides, CU and CU17 induced G2/M phase cell cycle arrest and p53-independent apoptosis in A549 cells. Both CU and CU17 down-regulated the expression of p53, p21, Bcl-2, and pERK1/2, but up-regulated Bax expression in this cell line. Although CU17 inhibited the growth of lung cancer cells less effectively than CU, it showed less toxicity than CU for non-cancer cells. Accordingly, CU17 is a promising agent for lung cancer treatment. Additionally, CU17 synergized the antiproliferative activity of Gem in A549 cells, indicating the possibility of employing CU17 as an adjuvant treatment to enhance the chemotherapeutic effect of Gem in lung cancer.     

HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing

Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.     

Structure-based design of potent non-peptide MDM2 inhibitors

A successful structure-based design of a class of non-peptide small-molecule MDM2 inhibitors targeting the p53-MDM2 protein-protein interaction is reported. The most potent compound 1d binds to MDM2 protein with a Ki value of 86 nM and is 18 times more potent than a natural p53 peptide (residues 16-27). Compound 1d is potent in inhibition of cell growth in LNCaP prostate cancer cells with wild-type p53 and shows only a weak activity in PC-3 prostate cancer cells with a deleted p53. Importantly, 1d has a minimal toxicity to normal prostate epithelial cells. Our studies provide a convincing example that structure-based strategy can be employed to design highly potent, non-peptide, cell-permeable, small-molecule inhibitors to target protein-protein interaction, which remains a very challenging area in chemical biology and drug design.     

Involvement of Heat-Shock Protein 70 and P53 Proteins in Attenuation of UVC-lnduced Apoptosis by Thermal Stress in Hepatocellular Carcinoma Cells

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. In this study, we found that a prior exposure to mild stress (42 degrees C) can protect HepG2 (p53+/+) cells from a subsequent UVC-induced apoptosis determined by DNA fragmentation and ratio of sub-G1 peak, but no heat-enhanced protection was found in Hep3B (p53-/-) cells. Although a similar inductive pattern of HSP70 protein and mRNA was detected in the two cell lines under thermal stress, the effect of thermal stress on UVC-induced apoptosis in HepG2 and Hep3B cells was obviously different. Overexpression of HSP70 by transient transfection of HSP70 expression vector in HepG2 cells significantly inhibited UVC-induced cell death; however, this inhibitory effect did not occur in transfected-Hep3B cells. Treatment of HepG2 cells with p53-specific antisense oligonucleotide could effectively block the antiapoptotic effect of thermal stress on UVC-induced apoptosis and increase of intracellular wild-type p53 protein by transfecting wtp53 expression plasmid into Hep3B cells yielded more resistance to UVC irradiation after prior thermal stress exposure. The results reveal an involvement of p53 in the antiapoptotic effect of thermal stress on UVC irradiation. Finally, a p53 protein increase was detected in UVC-treated HepG2 cells and could be coimmunoprecipitated with HSP70 after a thermal stress treatment. Prolonged p53 binding activity and enhanced expression of p53-controlled genes such as G1 arrest and DNA damage 45 and wild-type p53 activation factor 1/Cdk-interacting protein 1 by thermal stress are also observed in UVC-irradiated HepG2 cells. Based on these results, we propose that the antiapoptotic effect of thermal stress is mediated by increasing HSP70 and modulating intracellular p53 function.     

Gene network and canonical pathway analysis in prostate cancer: a microarray study

The molecular mechanism playing a role in the development of prostate cancer (PCA) is not well defined. We decided to determine the changes in gene expression in PCA tissues and to compare them to those in non-cancerous samples. Prostate tissue samples were collected by needle biopsy from 21 PCA and 10 benign prostate hyperplasic (BPH) patients. Total RNA was isolated, cDNA was synthesized, and gene expression levels were determined by microarray method. In the progression to PCA, 738 up-regulated and 515 down-regulated genes were detected in samples. Analysis using Ingenuity Pathway Analysis (IPA) software revealed that 466 network and 423 functions-pathways eligible genes were up-regulated, and 363 network and 342 functions-pathways eligible genes were down-regulated. Up-regulated networks were identified around IL-1beta and insulin-like growth factor-1 (IGF-1) genes. The NFKB gene was centered around two up- and down-regulated networks. Up-regulated canonical pathways were assigned and four of them were evaluated in detail: acute phase response, hepatic fibrosis, actin cytoskeleton, and coagulation pathways. Axonal guidance signaling was the most significant down-regulated canonical pathway. Our data provide not only networks between the genes for understanding the biologic properties of PCA but also useful pathway maps for future understanding of disease and the construction of new therapeutic targets.     

Transactivation of bad by vorinostat-induced acetylated p53 enhances doxorubicin-induced cytotoxicity in cervical cancer cells

Vorinostat (VOR) has been reported to enhance the cytotoxic effects of doxorubicin (DOX) with fewer side effects because of the lower DOX dosage in breast cancer cells. In this study, we investigated the novel mechanism underlying the synergistic cytotoxic effects of VOR and DOX co-treatment in cervical cancer cells HeLa, CaSki and SiHa cells. Co-treatment with VOR and DOX at marginal doses led to the induction of apoptosis through caspase-3 activation, poly (ADP-ribose) polymerase cleavage and DNA micronuclei. Notably, the synergistic growth inhibition induced by the co-treatment was attributed to the upregulation of the pro-apoptotic protein Bad, as the silencing of Bad expression using small interfering RNA (siRNA) abolished the phenomenon. As siRNA against p53 did not result in an increase in acetylated p53 and the consequent upregulation of Bad, the observed Bad upregulation was mediated by acetylated p53. Moreover, a chromatin immunoprecipitation analysis showed that the co-treatment of HeLa cells with VOR and DOX increased the recruitment of acetylated p53 to the bad promoter, with consequent bad transactivation. Conversely, C33A cervical cancer cells containing mutant p53 co-treated with VOR and DOX did not exhibit Bad upregulation, acetylated p53 induction or consequent synergistic growth inhibition. Together, the synergistic growth inhibition of cervical cancer cell lines induced by co-treatment with VOR and DOX can be attributed to the upregulation of Bad, which is induced by acetylated p53. These results show for the first time that the acetylation of p53, rather than histones, is a mechanism for the synergistic growth inhibition induced by VOR and DOX co-treatments.     

Lactucin,a Bitter Sesquiterpene from Cichorium intybus,Inhibits Cancer Cell Proliferation by Downregulating the MAPK and Central Carbon Metabolism Pathway

Lung cancer, especially adenocarcinoma, is the second most occurring and highest fatality-causing cancer worldwide. Many natural anticancer compounds, such as sesquiterpene lactones (SLs), show promising anticancer properties. Herein, we examined Lactucin, an SL from the plant Cichorium intybus, for its cytotoxicity, apoptotic-inducing, cell cycle inhibiting capacity, and associated protein expression. We also constructed a biotinylated Lactucin probe to isolate interacting proteins and identified them. We found that Lactucin stops the proliferation of A549 and H2347 lung adenocarcinoma cell lines while not affecting normal lung cell MRC5. It also significantly inhibits the cell cycle at G₀/G₁ stage and induces apoptosis. The western blot analysis shows that Lactucin downregulates the MAPK pathway, cyclin, and cyclin-dependent kinases, inhibiting DNA repair while upregulating p53, p21, Bax, PTEN, and downregulation of Bcl-2. An increased p53 in response to DNA damage upregulates p21, Bax, and PTEN. In an activity-based protein profiling (ABPP) analysis of A549 cell’s protein lysate using a biotinylated Lactucin probe, we found that Lactucin binds PGM, PKM, and LDHA PDH, four critical enzymes in central carbon metabolism in cancer cells, limiting cancer cells in its growth; thus, Lactucin inhibits cancer cell proliferation by downregulating the MAPK and the Central Carbon Metabolism pathway.     

Cholesterol secosterol aldehydes induce amyloidogenesis and dysfunction of wild-type tumor protein p53

Epidemiologic and clinical evidence points to an increased risk for cancer when coupled with chronic inflammation. However, the molecular mechanisms that underpin this interrelationship remain largely unresolved. Herein we show that the inflammation-derived cholesterol 5,6-secosterol aldehydes, atheronal-A (KA) and -B (ALD), but not the polyunsaturated fatty acid (PUFA)-derived aldehydes 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo red dyes but cannot bind to a consensus DNA sequence. Treatment of lung carcinoma cells with KA and ALD leads to a loss of function of extracted p53, as determined by the analysis of extracted nuclear protein and in activation of p21. Our results uncover a plausible chemical link between inflammation and cancer and expand the already pivotal role of p53 dysfunction and cancer risk.     

p21Cip/WAF1 activation is an important factor for the ERK pathway dependent anti-proliferation of colorectal cancer cells

p21Cip/WAF1, an important regulator of cell proliferation, is induced by both p53- and extracellular signal regulated kinase (ERK) pathways. The induction of p21Cip/WAF1 occurs by prolonged activation of the ERKs caused by extracellular stimuli, such as zinc. However, not all the cells appeared to respond to ERK pathway dependent p21Cip/WAF1 induction. Here we investigated the cause of such difference using colorectal cancer cells. p21Cip/WAF1 induction and concomitant reduction of bromodeoxyuridine (BrdU) incorporation were observed by zinc treatment within HT-29 and DLD-1. However, HCT-116 cells with high endogenous p21Cip/WAF1 levels did not show any additional increment of p21Cip/WAF1 levels by zinc treatment and did maintain high BrdU incorporation level. The p21Cip/WAF1 induction by zinc depended upon prolonged activation of extracellular signal regulated kinase (ERK) was not observed in HCT-116 cells. The percentage of BrdU positive cells was 50% higher in p21Cip/WAF1 -/- HCT-116 cells compared to p21Cip/WAF1 +/+ HCT- 116 cells, and no cells induced p21Cip/WAF1 incorporated BrdU in its nucleus, yet confirming the importance of p21Cip/WAF1 induction in anti- proliferation. These results again support that p21Cip/WAF1 induction is a determinant in the regulation of colonic proliferation by the ERK pathway.     

Role of p21CIP1 as a determinant of SC-560 response in human HCT116 colon carcinoma cells

SC-560, a structural analogue of celecoxib, induces growth inhibition in a wide range of human cancer cells in a cyclooxygenase (COX)-independent manner. Since SC-560 suppresses the growth of cancer cells mainly by inducing cell cycle arrest, we sought to examine the role of p21CIP1, a cell cycle regulator protein, in the cellular response against SC-560 by using p21(+/+) and p21(-/-) isogenic HCT116 colon carcinoma cells. In HCT116 (p21(+/+)) cells, SC-560 dose-dependently induced growth inhibition and cell cycle arrest at the G1 phase without significant apoptosis induction. SC-560-induced cell cycle arrest was accompanied by upregulation of p21CIP1. However, the extent of SC-560-induced accumulation at the G1 phase was approximately equal in the p21(+/+) and the p21(-/-) cells. Nonetheless, the growth inhibition by SC-560 was increased in p21(-/-) cells than p21(+/+)cells. SC-560-induced reactive oxygen species (ROS) generation did not differ between p21(+/+) and p21(-/-) cells but the subsequent activation of apoptotic caspase cascade was more pronounced in p21(-/-) cells compared with p21(+/+) cells. These results suggest that p21CIP1 blocks the SC-560-induced apoptotic response of HCT116 cells. SC-560 combined with other therapy that can block p21 CIP1 expression or function may contribute to the effective treatment of colon cancer.     

Protective Effects of L-Theanine on IPEC-J2 Cells Growth Inhibition Induced by Dextran Sulfate Sodium via p53 Signaling Pathway

L-theanine is a nonprotein amino acid found in tea leaves and has been widely used as a safe food additive in beverages or foods because of its varied bioactivities. The aim of this study was to reveal the in vitro gastrointestinal protective effects of L-theanine in DSS-induced intestinal porcine enterocyte (IPEC-J2) cell models using molecular and metabolic methods. Results showed that 2.5% dextran sulfate sodium (DSS) treatment inhibited the cell proliferation of IPEC-J2 and blocked the normal operation of the cell cycle, while L-theanine pretreatment significantly preserved these trends to exert protective effects. L-theanine pre-treatment also up-regulated the EGF, CDC2, FGF2, Rb genes and down-regulated p53, p21 proliferation-related mRNA expression in DSS-treated cells, in accompany with p53 signaling pathway inhibition. Meanwhile, metabolomics analysis revealed that L-theanine and DSS treated IPEC-J2 cells have different metabolomic profiles, with significant changes in the key metabolites involved in pyrimidine metabolism and amino acid metabolism, which play an important role in nucleotide metabolism. In summary, L-theanine has a beneficial protection in DSS-induced IPEC-J2 cells via promoting proliferation and regulating metabolism disorders.     

A Two‐Stage Dissociation System for Multilayer Imaging of Cancer Biomarker‐Synergic Networks in Single Cells

The monitoring of cancer biomarkers is crucial to the early detection of cancer. However, a limiting factor in biomarker analysis is the ability to obtain the multilayered information of various biomarker molecules located at different parts of cells from the plasma membrane to the cytoplasm. A two‐stage dissociation nanoparticle system based on multifunctionalized polydopamine‐coated gold nanoparticles (Au@PDA NPs) is reported, which allows for the two‐stage imaging of cancer biomarkers in single cells. We demonstrate the feasibility of this strategy on sialic acids (SAs), p53 protein, and microRNA‐21 (miRNA‐21) in MCF‐7 breast cancer cells by two custom‐built probes. Furthermore, the multicolor fluorescence information extracted is used for the monitoring of biomarker expression changes under different drug combinations, which allows us to investigate the complex interactions between various cancer biomarkers and to describe the cancer biomarker‐synergic networks in single cells.     

The p53‐Dependent Expression of Frataxin Controls 5‐Aminolevulinic Acid‐Induced Accumulation of Protoporphyrin IX and Photo‐Damage in Cancerous Cells

Mitochondrial frataxin is involved in various functions such as iron homeostasis, iron–sulfur cluster biogenesis, the protection from oxidative stress and apoptosis and acts as a tumor suppressor protein. We now show that the expression of frataxin is stimulated in a p53‐dependent manner and prove that frataxin is a direct p53 target gene by showing that the p53‐responsive element in the promoter of the mouse frataxin gene is bound by p53. The bacterial expression of human frataxin stimulated maturation of human ferrochelatase, which catalyzes the insertion of iron into protoporphyrin at the last step of heme biosynthesis. Overexpression of frataxin in human cancer A431 and HeLa cells lowered 5‐aminolevulinic acid(ALA)‐induced accumulation of protoporphyrin and induced resistance to ALA‐induced photo‐damage, whereas p53 silencing with siRNA in non tumor HEK293T cells down‐regulated the expression of frataxin and increased the accumulation of protoporphyrin. Thus, the decrease of the expression of frataxin unregulated by p53 in tumor cells enhances ALA‐induced photo‐damage, by down‐regulation of mitochondrial functions.