脆性组氨酸三联体基因研究进展

脆性组氯酸三联体（FHIT）基因定位于染色体的3p14．2,经细胞生物学、肿瘤分子生物学研究,及转基因、基因敲除技术等实验证实其为抑癌基因。FHIT主要通过某种信号途径诱导凋亡,从而抑制肿瘤细胞的增殖。研究认为,FHIT通过FHIT-底物复合物产生抑癌作用。越来越多的研究结果表明FHIT在肿瘤的预防和治疗中具有很好的应用前景。     

TRAIL‐induced apoptosis of FHIT‐negative lung cancer cells is inhibited by FHIT re‐expression

Fragile histidine triad (FHIT) is a tumor suppressor gene whose allelic loss is associated to a number of human cancers. FHIT protein acts as a diadenosine oligophosphate hydrolase, but its tumor suppressive activity appears as independent from its enzymatic activity. Tumor necrosis factor (TNF)‐related apoptosis inducing ligand (TRAIL) can induce apoptosis in the FHIT‐negative non‐small lung cancer cell line Calu‐1. We generated four FHIT‐inducible Calu‐1 cell clones and demonstrated that FHIT expression was able to protect cells from TRAIL‐induced apoptosis, without affecting TRAIL‐receptors surface expression. FHIT‐specific small interference RNA transfection of SV40‐immortalized normal bronchial BEAS cells that show levels of FHIT protein comparable to those of normal bronchial cells, resulted in a significant increase of TRAIL‐induced apoptosis. Of note, suramin‐mediated inhibition of FHIT enzymatic activity also enhanced TRAIL‐induced apoptosis. We conclude that FHIT expression in lung cancer cells is protective from TRAIL‐induced apoptosis. Our data suggest that FHIT exerts this protective effect downstream TRAIL‐receptors and likely requires its dinucleoside‐triphosphate hydrolase activity. As TRAIL represents in the near future a good candidate for death ligands‐based anticancer therapy, its potential therapeutic use should be envisaged as preliminary to molecular genetics interventions or drug‐induced epigenetic modulations aimed to restoring FHIT gene expression levels in non‐small cells lung tumors. J. Cell. Physiol. 220: 492–498, 2009. © 2009 Wiley‐Liss, Inc.     

TRAIL induces apoptosis but not necroptosis in colorectal and pancreatic cancer cells preferentially via the TRAIL-R2/DR5 receptor

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that can trigger apoptosis in many types of human cancer cells via engagement of its two pro-apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). TRAIL can also activate several other signaling pathways such as activation of stress kinases, canonical NF-κB signaling and necroptosis. Though both receptors are ubiquitously expressed, their relative participation in TRAIL-induced signaling is still largely unknown. To analyze TRAIL receptor-specific signaling, we prepared Strep-tagged, trimerized variants of recombinant human TRAIL with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, we examined the contribution of individual pro-apoptotic receptors to TRAIL-induced signaling pathways. We found that in TRAIL-resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeds comparably via both DR4- and DR5-activated signaling. TRAIL-induced apoptosis, enhanced by the inhibitor of the Bcl-2 family ABT-737, or by the translation inhibitor homoharringtonine, proceeded in both cell lines predominantly via the DR5 receptor. ShRNA-mediated downregulation of DR4 or DR5 receptors in HT-29 cells also pointed to a stronger contribution of DR5 in TRAIL-induced apoptosis. In contrast to apoptosis, necroptotic signaling was activated similarly by both DR4- or DR5-specific ligands. Activation of auxiliary signaling pathways involving NF-κB or stress kinases proceeded under apoptotic conditions mainly in a DR5-dependent manner, while these signaling pathways were during necroptosis similarly activated by either of these ligands. Our study provides the first systematic insight into DR4 ?/DR5-specific signaling in colorectal and pancreatic cancer cells.     

Death receptors: Targets for cancer therapy

Apoptosis is the cell's intrinsic program to death, which plays an important role in physiologic growth control and homeostasis. Apoptosis can be triggered by death receptors (DRs), without any adverse effects. DRs are the members of tumor necrosis factor (TNF) receptor superfamily, known to be involved in apoptosis signaling, independent of p53 tumor-supressor gene. Selective triggering of DR-mediated apoptosis in cancer cells is a novel approach in cancer therapy. So far, the best characterized DRs are CD95 (Fas/Apo1), TNF-related apoptosis-inducing ligand receptor (TRAILR) and tumor necrosis factor receptor (TNFR). Among these, TRAILR is emerging as most promising agent for cancer therapy, because it induces apoptosis in a variety of tumor and transformed cells without any toxicity to normal cells. TRAIL treatment in combination with chemotherapy or radiotherapy enhances TRAIL sensitivity or reverses TRAIL resistance by regulating downstream effectors. This review covers the current knowledge about the DRs, summarizes main signaling in DRs and also summarizes the preclinical approaches of these DRs in cancer therapy.     

Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas.

BACKGROUND: Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes. Loss of FHIT function may be important in the development and/or progression of various types of cancer. MATERIALS AND METHODS: We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC). Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, [3H]-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections. RESULTS: The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation. Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues. The observed changes increased with progression of these lesions. We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci. We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC). The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months. FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant. Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05). CONCLUSIONS: Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation. Aberrant FHIT gene could be a prognostic marker in lung cancer.     

The farnesyltransferase inhibitor lonafarnib induces CCAAT/enhancer-binding protein homologous protein-dependent expression of death receptor 5, leading to induction of apoptosis in human cancer cells

Pre-clinical studies have demonstrated that farnesyltransferase inhibitors (FTIs) induce growth arrest or apoptosis in various human cancer cells independently of Ras mutations. However, the underlying mechanism remains unknown. Death receptor 5 (DR5) is a pro-apoptotic protein involved in mediating the extrinsic apoptotic pathway. Its role in FTI-induced apoptosis has not been reported. In this study, we investigated the modulation of DR5 by the FTI lonafarnib and the involvement of DR5 up-regulation in FTI-induced apoptosis. Lonafarnib activated caspase-8 and its downstream caspases, whereas the caspase-8-specific inhibitor benzyloxycarbonyl-Ile-Glu(methoxy)-Thr-Asp(methoxy)-fluoromethyl ketone or small interfering RNA abrogated lonafarnib-induced apoptosis, indicating that lonafarnib induces caspase-8-dependent apoptosis. Lonafarnib up-regulated DR5 expression, increased cell-surface DR5 distribution, and enhanced tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Overexpression of a dominant-negative Fas-associated death domain mutant or silencing of DR5 expression using small interfering RNA attenuated lonafarnib-induced apoptosis. These results indicate a critical role of the DR5-mediated extrinsic apoptotic pathway in lonafarnib-induced apoptosis. By analyzing the DR5 promoter, we found that lonafarnib induced a CCAAT/enhancer-binding protein homologous protein (CHOP)-dependent transactivation of the DR5 promoter. Lonafarnib increased CHOP expression, whereas silencing of CHOP expression abrogated lonafarnib-induced DR5 expression. These results thus indicate that lonafarnib induces CHOP-dependent DR5 up-regulation. We conclude that CHOP-dependent DR5 up-regulation contributes to lonafarnib-induced apoptosis.     

Molecular genetic alterations of FHIT and p53 genes in benign and malignant thyroid gland lesions

Several oncogenes and tumor-suppressor genes are involved either as early or late event in thyroid gland carcinogenesis. Human FHIT (fragile histidine triad) gene is highly conserved gene whose loss of function may be important in the development and/or progression of various types of cancer. We undertook this study to analyze FHIT and p53 gene status in different benignant and malignant thyroid tumors. Status of these genes as well as intensity of apoptosis was analyzed in tumor tissues by molecular genetic methods, immunohistochemistry, and FACS-scan analysis. The majority of the malignant thyroid cancers displayed aberrant expression of FHIT gene, concominant with p53 gene inactivation. This is followed by low rate of apoptosis, which may be important in the development and/or progression of thyroid cancer. We found higher incidence of p53 mutation and aberrant processing of FHIT mRNA in malignant tumors (papillary, follicular, medullary and anaplastic carcinomas) and in those tumors with distant metastasis. The growth of p53(-)/FHIT(-) follicular carcinoma of human origin was much faster in nude mice than p53(+)/FHIT(+) follicular carcinoma, and mice had shorter survival rate. Our results show a correlation between aberrant FHIT and p53 expression, low rate of apoptosis, and malignancy. Concomitant aberration of FHIT gene and p53 could be responsible for development of highly malignant types of thyroid cancer and may be considered as a prognostic marker for these tumors.     

LINC00184 plays an oncogenic role in non-small cell lung cancer via regulation of the miR-524-5p/HMGB2 axis

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. We aimed to investigate the role of LINC00184 in NSCLC. Migration, proliferation and invasion of NSCLC cells were analysed using the wound healing assay, cell counting kit-8 assay and transwell assay, respectively. Apoptosis and cell cycle were assessed using flow cytometry. Online bioinformatics tools were utilized to predict downstream microRNAs (miRNA) or genes related to LINC00184 expression. The RNA pull-down experiment and luciferase reporter assay were performed to verify the predictions thereof. LINC00184, miR-524-5p, and high mobility group 2 protein (HMGB2) expression levels in NSCLC tissues and cell lines were detected using quantitative real-time polymerase chain reaction. An NSCLC mouse model was constructed for in vivo experiments. LINC00184 overexpression was observed in NSCLC tissues and cell lines and was found to be correlated with poor prognosis. LINC00184 knockdown inhibited cell proliferation, migration and invasion, induced cell cycle arrest and accelerated apoptosis in NSCLC cell lines. LINC00184 suppressed tumour growth and proliferation in NSCLC mouse models and directly targeted the miR-524-5p/HMGB2 axis. Moreover, the expression levels of LINC00184 and HMGB2 were negatively correlated with miR-524-5p expression, whereas LINC00184 expression was positively correlated with HMGB2 expression. LINC00184 affected the cell cycle, proliferation, apoptosis, migration and invasion in NSCLC via regulation of the miR-524-5p/HMGB2 axis.     

Docosahexaenoic acid induces apoptosis in lung cancer cells by increasing MKP-1 and down-regulating p-ERK1/2 and p-p38 expression

Different agents able to modulate apoptosis have been shown to modify the expression of the MAP-kinase-phosphatase-1 (MKP-1). The expression of this phosphatase has been considered a potential positive prognostic factor in lung cancer, and smoke was shown to reduce the levels of MKP-1 in ferret lung. Our aim was to assess whether the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), known to inhibit the growth of several cancer cells mainly inducing apoptosis, may exert pro-apoptotic effect in lung cancer cells by modifying MKP-1 expression. We observed that DHA increased MKP-1 protein and mRNA expression and induced apoptosis in different lung cancer cell lines (mink Mv1Lu adenocarcinoma cells, human A549 adenocarcinoma and human BEN squamous carcinoma cells). We inhibited the pro-apoptotic effect of DHA by treating the cells with the phosphatase inhibitor Na₃VO₄ or by silencing the MKP-1 gene with the specific siRNA. This finding demonstrated that the induction of apoptosis by DHA involved a phosphatase activity, specifically that of MKP-1. DHA reduced also the levels of the phosphorylated MAP-kinases, especially ERK1/2 and p38. Such an effect was not observed when the MKP-1 gene was silenced. Altogether, the data provide evidence that the DHA-induced overexpression of MKP-1 and the resulting decrease of MAP-kinase phosphorylation by DHA may underlie the pro-apoptotic effect of this fatty acid in lung cancer cells. Moreover, they support the hypothesis that DHA may exert chemopreventive action in lung cancer.     

Signaling Crosstalk of FHIT,p53, and p38 in etoposide-induced apoptosis in MCF-7 cells

Fragile histidine trail (FHIT) is a tumor suppressor in response to DNA damage which has been deleted in various tumors. However, the signaling mechanisms and interactions of FHIT with regard to apoptotic proteins including p53 and p38 in the DNA damage-induced apoptosis are not well described. In the present study, we used etoposide-induced DNA damage in MCF-7 as a model to address these crosstalks. The time course study showed that the expression of FHIT, p53, and p38MAPK started after 1 hour following etoposide treatment. FHIT overexpression led to increase p53 expression, p38 activation, and augmented apoptosis following etoposide-induced DNA damage compared to wild-type cells. However, FHIT knockdown blocked p53 expression, delayed p38 activation, and completely inhibited etoposide-induced apoptosis. Inhibition of p38 activity prevented induction of p53, FHIT, and apoptosis in this model. Thus, activation of p38 upon etoposide treatment leads to increase in FHIT and p53 expression. In p53 knockdown MCF-7, the FHIT induction was hampered but p38 activation was induced in lower doses of etoposide. In p53 knockdown cells, inhibition of p38 induced FHIT expression and apoptosis. Our data demonstrated that the exposure of MCF-7 cells to etoposide increases apoptosis through a mechanism involving the activation of the p38-FHIT-p53 pathway. Moreover, our findings suggest signaling interaction for these pathways may represent a promising therapy for breast cancer.     

LukS-PV induces cell cycle arrest and apoptosis through p38/ERK MAPK signaling pathway in NSCLC cells

Non-small-cell lung cancer (NSCLC) accounts for nearly 85% of lung cancer cases. LukS-PV, one of the two components of Panton-Valentine leucocidin (PVL), is produced by Staphylococcus aureus. The present study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) lines (THP-1 and HL-60). However, the role of LukS-PV in NSCLC is unclear. In this study, we treated NSCLC cell lines A549 and H460 and a normal lung cell line, 16HBE, with LukS-PV and investigated the biological roles of LukS-PV in NSCLC. Cells were treated with varying concentrations of LukS-PV and cell viability was evaluated by CCK8 and EdU assay. Flow cytometry was used to detect cell apoptosis and analyze the cell cycle, and the expression of apoptosis and cell cycle-associated proteins and genes were identified by western blotting analysis and qRT-polymerase chain reaction, respectively. We found that LukS-PV inhibited the proliferation of NSCLC cells but had little cytotoxicity in normal lung cells. LukS-PV induced NSCLC cell apoptosis and increased the BAX/BCL-2 ratio, triggering S-phase arrest in A549 and H460 cells while increasing P21 expression and decreasing CDK2, cyclin D1, and cyclin A2 expression. We also observed increased P-p38 and P-ERK in NSCLC cells treated with LukS-PV. Treatment of NSCLC with LukS-PV combined with p38 and ERK inhibitors reversed the pro-apoptotic and pro-cell cycle arrest effects of LukS-PV. Overall, these findings indicate that LukS-PV has anti-tumor effects in NSCLC and may contribute to the development of anti-cancer agents.     

The relative contribution of pro-apoptotic p53-target genes in the triggering of apoptosis following DNA damage in vitro and in vivo

The p53 pathway displays a large degree of redundancy in the expression of a number of pro-apoptotic mechanisms following DNA damage that, among others, involves increased expression of several pro-apoptotic genes through transactivation. Spatial and temporal cellular contexts contribute to the complexity of the regulation of apoptosis, hence different genes may show a cell- and tissue-dependent specificity with regard to the regulation of cell death and act in concert or show redundancy with one and another. We used siRNA technology to assess the effect of multiple ablations of documented pro-apoptotic p53 target genes (PPG) in the colorectal cancer cell line HCT116 and generated mice deficient in both of the extrinsic and intrinsic PPGs genes Dr5 and Puma following treatment with chemotherapeutics and ionizing radiation. DR5, Fas, Bax, Bad, Puma and Bnip3L were induced by 5-FU and adriamycin (ADR) in HCT116 cells in a p53-dependent manner. The resulting caspase 3/7 activity in HCT116 cells following treatment were suppressed by ablated expression of the PPGs in the extrinsic as well as the intrinsic pathway. To our surprise, knocking-down any of the PPGs concomitantly with DR5 did not further inhibit caspase 3/7 activity whereas inhibiting DR5-expression in HCT116Bax knockdown (kd) and HCT116Fas kd did, suggesting that these genes act downstream or in synergy with DR5. This was supported by our in vivo observations, since Puma and Dr5 were equally efficient in protecting cells of the spleen from sub-lethal radiation-induced apoptosis but less effective compared with irradiated p53^-/- mice. To our surprise, Dr5^-/-; Puma^-/- mice did not show additive protection from radiation-induced apoptosis in any of the investigated organs. Our data indicates that the intrinsic pathway may rely on extrinsic signals to promote cell death in a cell- and tissue-dependent manner following DNA damage. Furthermore, p53 must rely on mechanisms independent of DR5 and PUMA to initiate apoptosis following γ-radiation in the spleen and thymus in vivo.     

p53siRNA therapy reduces cell proliferation, migration and induces apoptosis in triple negative breast cancer cells

p53 protein is probably the best known tumor suppressor. Earlier reports proved that human breast cancer cells expressing mutant p53 displayed resistance to apoptosis. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block p53 expression, as well as its subsequent effect on cell growth, apoptosis and migration on a triple negative human breast cancer cell line (Hs578T). p53siRNA significantly reduced cell index (CI) compared to the control and we observed an inhibition of cellular migration in the interval of time between 0 and 30 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. Also, by using PCR-array technology, a panel of 84 key genes involved in apoptosis was investigated. Our studies indicate that the knockdown of p53 expression by siRNA modulates several genes involved in cell death pathways and apoptosis, showing statistically significant gene expression differences for 22 genes, from which 18 were upregulated and 4 were downregulated. The present research also emphasizes the important role of BCL-2 pro-apoptotic family of genes (Bim, Bak, and Bax) in activating apoptosis and reducing cell proliferation by p53siRNA treatment. Death receptors cooperate with BCL-2 pro-apoptotic genes in reducing cell proliferation. The limited success may be due to the activation of the antiapoptotic gene Mcl-1, and it may be associated with the resistance of triple negative breast cancer cells to cancer treatment. Thus, targeting p53siRNA pathways using siRNA may serve as a promising therapeutic strategy for the treatment of breast cancers.     

Alpha-tocopheryl succinate induces DR4 and DR5 expression by a p53-dependent route: implication for sensitisation of resistant cancer cells to TRAIL apoptosis

We evaluated the ability of alpha-tocopheryl succinate (alpha-TOS) to sensitise TRAIL-resistant malignant mesothelioma (MM) cells to TRAIL-induced apoptosis. We show that alpha-TOS activates expression of DR4/DR5 in a p53-dependent manner and re-establishes sensitivity of resistant MM cells to TRAIL-mediated apoptosis, as documented in p53wt MM cells but not in their p53null counterparts. MM cells selected for TRAIL resistance expressed low cell surface levels of DR4 and DR5. Treatment with sub-lethal doses of alpha-TOS restored expression of DR4 and DR5. The ability of alpha-TOS to modulate expression of pro-apoptotic genes may play a role in sensitisation of tumour cells to immunological stimuli.