Pharmacological inhibition of fatty acid synthase activity produces both cytostatic and cytotoxic effects modulated by p53

Fatty acid synthetic metabolism is abnormally elevated in tumor cells, and pharmacological inhibitors of the anabolic enzyme fatty acid synthase (FAS), including the natural product cerulenin and the novel synthetic compound c75, are selective inhibitors of tumor cell growth. We have recently reported that these two FAS inhibitors both produce rapid, potent inhibition of DNA replication and S-phase progression in human cancer cells, as well as apoptotic death. Here we report an additional characterization of the cellular response to FAS inhibition. RKO colon carcinoma cells were selected for study because they undergo little apoptosis within the first 24 h after FAS inhibition. Instead, RKO cells exhibited a biphasic stress response with a transient accumulation in S and G2 at 4 and 8 h that corresponds to a marked reduction in cyclin A- and B1-associated kinase activities, and then by accumulation of p53 and p21 proteins at 16 and 24 h and growth arrest in G1 and G2. The response of RKO cells to FAS inhibition resembled a genotoxic stress response, but DNA damage did not appear to be an important downstream effect of FAS inhibition, because none was detected using the single cell gel electrophoresis assay (comet assay) to assess DNA damage. p53 function is probably important in protecting RKO cells from FAS inhibition because, similar to many other tumor lines, RKO cells expressing a dominant negative mutant p53 gene underwent extensive apoptosis within 24 h after FAS inhibition. Sensitization of cells to FAS inhibitors by the loss of p53 raises the possibility that these agents may be clinically useful against malignancies carrying p53 mutations. Whereas induction of apoptosis appeared related to accumulation of the substrate, malonyl-CoA, after FAS inhibition, the cytostatic effects were independent of malonyl-CoA accumulation and may have resulted from product depletion.     

Growth arrest and apoptosis of the human hepatocellular carcinoma cell line BEL-7402 induced by melittin

OBJECTIVE: The aim of this study was to investigate the cellular effects of melittin on the growth and apoptosis of human hepatocellular carcinoma (HCC) cells and to provide the molecular mechanism for potential application of a recombinant adenovirus carrying the melittin gene (Ad-rAFP-Mel) in the treatment of liver cancer. METHODS: Human HCC cells (BEL-7402) were infected with Ad-rAFPMel at different times. In vitro cell growth was determined by MTT assay. Cellular apoptosis was evaluated quantitatively and qualitatively by phase-contrast microscopy, transmission electron microscopy, DNA ladder electrophoresis, TUNEL staining and flow cytometry. RESULTS: Ad-rAFP-Mel infection had an inhibitory effect on the proliferation of BEL-7402 cells. The morphological changes of apoptosis were confirmed by microscopy and DNA electrophoresis. The ultrastructural characteristics of apoptotic cells, such as chromatin condensation and nuclear fragmentation, were also observed by electron microscopy in the Ad-rAFP-Mel-infected cells. Ad-rAFPMel infection markedly induced cellular apoptosis, and Fas expression on Bel-7402 cells infected by Ad-rAFPMel was up-regulated. CONCLUSION: The fact that melittin can induce apoptosis of the HCC cell line BEL-7402 leads us to consider adenovirus-mediated delivery of melittin as a promising approach for the treatment of HCC. However, the underlying mechanism needs to be further investigated.     

缺氧对人肝癌HepG2细胞凋亡及p38MAPK通路的影响

目的:探讨缺氧对人肝癌HepG2细胞凋亡及p38MAPK通路的影响。方法:用三气培养箱培养肝癌细胞HepG2,建立缺氧细胞模型,CCK-8 法检测HepG2细胞的增殖率,流式细胞术检测细胞凋亡率,Real-time PCR分析p38MAPK mＲNA的表达,Western blot法检测p38MAPK及p-p38MAPK蛋白的表达变化。结果:缺氧可抑制人肝癌HepG2细胞的增殖,抑制细胞凋亡（P<0.05）;缺氧处理人肝癌HepG2细胞后,p38MAPK mＲNA的表达量下调(P<0.05);Western blot结果显示缺氧可下调p38MAPK、p-p38MAPK蛋白的表达（P<0.05）。结论:缺氧通过抑制p38MAPK通路抑制人肝癌HepG2细胞凋亡。     

The Rho kinase inhibitor fasudil is involved in p53-mediated apoptosis in human hepatocellular carcinoma cells

Purpose

Rho kinase is an important factor in tumor progression. We demonstrated that Rho kinase-associated coil-containing protein kinase (ROCK) is expressed in hepatic tissues in hepatocellular carcinoma (HCC) and confirmed its roles in cell survival in HCC cells using the ROCK inhibitor, fasudil.

Methods

ROCK protein levels were estimated in hepatic tissues with HCC compared with healthy liver tissues or hepatic hemangioma tissues using immunohistochemistry. Furthermore, HepG2 and Huh7 cells were cultured with ROCK inhibitor, fasudil for 24?h in vitro. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, and apoptotic cells were detected by cell death ELISA. The expression apoptosis-related proteins were analyzed using Western blotting.

Results

Fasudil significantly decreased cell proliferation and induced apoptosis mediated by increases in p53, Bax, caspase-9, and caspase-3 in HepG2 and Huh7 cells. The induction of apoptosis was inhibited in HCC cells precultured with p53 decoy oligodeoxynucleotide.

Conclusion

These results suggest that ROCK inhibits the p53-mediated apoptosis pathway in HCC. Fasudil may thus be a beneficial approach to HCC therapy.     

Hibiscus polyphenol-rich extract induces apoptosis in human gastric carcinoma cells via p53 phosphorylation and p38 MAPK/FasL cascade pathway

In view of the continuing need for effective anticancer agents, and the association of diet with reduced cancer risk, edible plants are increasingly being considered as sources of anticancer drugs. Hibiscus sabdariffa Linne (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in the Sudan and Eastern Taiwan. Polyphenols had been demonstrated previously to possess antioxidative and antitumor promoting effects. In this study, investigations were conducted to examine the mechanism of the anticancer activity of H. sabdariffa L., Hibiscus polyphenol-rich extracts (HPE). Using HPLC assay, HPE was demonstrated to contain various polyphenols. HPE induced cell death of eight kinds of cell lines in a concentration-dependent manner. Among them human gastric carcinoma (AGS) cells were the most susceptible to HPE (0.95 mg/mL HPE inhibited its growth by 50%). Our results revealed that AGS cells underwent DNA fragmentation, and had an increase in the distribution of hypodiploid phase (apoptotic peak, 52.36%) after a 24-h treatment with HPE (2.0 mg/mL). This effect of HPE in AGS cells might be mediated via p53 signaling and p38 MAPK/FasL cascade pathway, as demonstrated by an increase in the phosphorylation of p53 and the usage of a specific p38 inhibitor, SB203580. Thus, our data present the first evidence of HPE as an apoptosis inducer in AGS cells and these findings may open interesting perspectives to the strategy in human gastric cancer treatment.     

p53 mutation, but not p53 overexpression, correlates with survival in head and neck squamous cell carcinoma.

Survival in squamous cell carcinoma of the head and neck (HNSCC) was compared with overexpression and mutation of the p53 gene. Archival tissue from 77 tumours was analysed for protein expression using immunohistochemistry (IHC) with the monoclonal antibody Do-7, and for the presence of mutation in exons 5-8 using single-stranded conformation polymorphism (SSCP), followed by DNA sequencing in SSCP-positive cases. p53 expression was scored as high (>70% nuclei stained) in 25 (32%) tumours, as intermediate (10-70% nuclei stained) in 19 (25%) tumours and as low (<10% nuclei stained) in 33 (43%) tumours. Twelve (18%) tumours exhibited gene mutation (ten missense and two nonsense mutations) and an additional five tumours contained changes that could not result in amino acid substitution or protein truncation. There was no correlation between gene expression and mutation, mutations being equally frequent in tumours with either high (4/25), intermediate (4/19) or low protein expression (4/33). Fifty-eight patients were eligible for survival analysis. There was a strong correlation between p53 mutation and cause-specific survival; median survival among mutated cases was 12.5 months compared with >160 months among non-mutated patients (P < 0.005). There was no correlation between p53 overexpression and survival. The results suggest that p53 mutation status is an important prognostic factor in HNSCC, and that IHC analysis of protein overexpression is an inadequate measure of gene mutation in these tumours.     

TGF-beta-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells

Transforming growth factor (TGF)-beta has been reported to exert growth inhibitory activity in normal epithelial cells whereas it induces cell proliferation and invasive phenotypes in advanced carcinomas. Our previous study showed that MCF10A, a spontaneously immortalized "normal" breast epithelial cell line, is resistant to TGF-beta-induced growth inhibition, suggesting that conversion of TGF-beta growth inhibitory signaling into an oncogenic pathway may occur at the early stage of tumor development/progression. To address this issue, we investigated the TGF-beta signaling pathway and its role in phenotypic transformation of MCF10A cells. TGF-beta treatment induced changes in the MCF10A cell morphology from cuboidal to an elongated spindle-like shape, accompanied with down-regulation of epithelial cell marker E-cadherin. TGF-beta treatment was sufficient to induce migrative and invasive phenotypes in these cells, an important phenotypic conversion during tumor progression. We also showed that TGF-beta treatment rapidly activated ERK-1/2 and p38 MAPK leading to upregulation of matrix metalloproteinase (MMP)-2 and MMP-9. Using chemical inhibitors and dominant negative mutants of MAPKs, we provide evidence that while both p38 MAPK and ERKs are required for TGF-beta-induced MCF10A cell migration and invasion, TGF-beta-induced MMP-2 and MMP-9 expression depends on p38 MAPK signaling, but is independent of ERK activity. This study demonstrates the roles of TGF-beta signaling pathways for induction of oncogenic signaling in preneoplastic human breast epithelial cells and will deepen our understanding of TGF-beta signaling in the progress of breast cancer.     

Lidamycin induces marked G2 cell cycle arrest in human colon carcinoma HT-29 cells through activation of p38 MAPK pathway

Lidamycin (LDM), a member of the enediyne antibiotic family, is presently undergoing phase I clinical trials in P.R. China. In this study, we investigated the mechanisms of LDM-induced cell cycle arrest in order to support its use in clinical cancer therapy. Using human colon carcinoma HT-29 cells, we observed that LDM induced G2 cell cycle arrest in a time- and dose-dependent manner. LDM-induced G2 arrest was associated with increasing phosphorylation of Chk1, Chk2, Cdc25C, Cdc2 and expression of Cdc2 and cyclin B1. In addition, cytoplasmic localization of cyclin B1 was also involved in LDM-induced G2 arrest. Moreover, we found that p38 MAPK pathway contributed to LDM-induced G2 arrest. Inhibition of p38 MAPK by its inhibitor SB203580 not only attenuated LDM-induced G2 arrest but also potentiated LDM-induced apoptosis, which was accompanied by decreasing phosphorylation of Cdc2 and increasing expression of FasL and phosphorylation of JNK. Finally, we demonstrated that cells at G1 phase were more sensitive to LDM. Together, our findings suggest that p38 MAPK signaling pathway is involved in LDM-induced G2 arrest, at least partly, and a combination of LDM with p38 MAPK inhibitor may represent a new strategy for human colon cancer therapy.     

NS-398, a selective cyclooxygenase 2 inhibitor,inhibited cell growth and induced cell cycle arrest in human hepatocellular carcinoma cell lines

Cyclooxygenase 2 (COX-2) has been suggested to be associated with liver carcinogenesis. Several reports have shown that NSAIDs inhibit the growth of hepatocellular carcinoma cell lines. There is little evidence of how COX-2 inhibitors regulate the proliferation of hepatocellular carcinoma cells or the mechanism involved. In our study, we investigated the growth-inhibitory mechanism of a selective COX-2 inhibitor, NS-398, in 4 hepatocellular carcinoma cell lines by studying cell growth, COX-2 and proliferating cell nuclear antigen (PCNA) expression, cell cycle distribution and the evidence of apoptosis. NS-398 inhibited the growth of all 4 cell lines in a time- and dose-dependent manner and the inhibitory effects were independent of the level of COX-2 protein expression. PCNA expression was downregulated by NS-398 in a dose-independent manner. NS-398 caused cell cycle arrest in the S phase with a reduction in cell numbers and cell accumulation in the G0/G1 phase, for all 4 cell lines. No evidence of apoptosis was observed in our present study. Our findings suggest that a selective COX-2 inhibitor might serve as an effective tool for the chemoprevention and treatment of hepatocellular carcinomas. A reduction in cell number in the S phase may be an important event in cell cycle arrest caused by NS-398 in hepatocellular carcinoma cell lines.     

Enhanced TRAIL sensitivity by p53 overexpression in human cancer but not normal cell lines

The cytotoxic ligand TRAIL is a promising anti-cancer agent that is entering into clinical trials. We previously identified a major subgroup of TRAIL resistant cancer cell lines with absent, or reduced DR4 expression containing a K441R polymorphism or harboring elevated levels of the caspase activation inhibitor FLIP. In the present study, we explored the use of a gene therapeutic approach utilizing p53, delivered by an adenovirus-p53 (Ad-p53) vector, which directly controls expression of the TRAIL receptor KILLER/DR5 in a panel of 8 cell lines including normal and TRAIL sensitive or resistant cancers. The functional status of the delivered p53 was monitored by detection of induced p21WAF1 expression by immunocytochemistry. In normal cells, which are TRAIL resistant, TRAIL did not reduce cell viability over and above the effect of Ad-p53 alone. All cancer cell lines were sensitive to Ad-p53 and up-regulated expression of the TRAIL receptor KILLER/DR5. TRAIL-resistant cancer cells became more sensitive to TRAIL at low Ad-p53 multiplicities of infection but TRAIL resistance was not completely overcome in one TRAIL-resistant cell line probably because of a high level of expression of FLIP. The results reveal that Ad-p53 induces the TRAIL receptor KILLER/DR5 and, like radiation or chemotherapy may effectively reverse TRAIL resistance.     

Phosphorylation of murine p53, but not human p53, by MAP kinase in vitro and in cultured cells highlights species-dependent variation in post-translational modification

The p53 tumour suppressor protein is tightly regulated by protein-protein association, protein turnover and a variety of post-translational modifications. Multisite phosphorylation plays a major role in activating and in finely tuning p53 function. The proline rich domain of murine p53 is a substrate for phosphorylation, in vitro and in cultured cells, by the p42ERK2 and p44ERK1 mitogen-activated protein (MAP) kinases. However, to date there have been no reports of attempts to determine whether p53 from any other species is a substrate for MAP kinase. In this paper we confirm that murine p53 is targeted by recombinant MAP kinase and by MAP kinases in extracts of both murine and human cells. In contrast, human p53 is not a substrate for recombinant MAP kinase nor are there any detectable levels of protein kinase activity in stimulated human cell extracts which phosphorylate the proline rich domain of human p53 in vitro. Finally, although stimulation of murine fibroblasts with o-tetradecanolylphorbol 13-acetate (TPA), an indirect activator of the MAP kinase pathway, leads to site-specific phosphorylation of murine p53, similar treatment of human fibroblasts and epithelial cells showed no significant changes in the phosphorylation pattern. These data are consistent with accumulating evidence that significant species-dependent differences exist in the post-translational modification of p53.     

Mutation of the p53 gene in a differentiated human thyroid carcinoma cell line, but not in primary thyroid tumours

The p53 gene has been implicated as a tumour suppressor, with mutations occurring in many carcinomas, such as colon, breast and lung. We have sequenced exons 5, 7 and 8 containing conserved gene regions in the only available differentiated thyroid follicular carcinoma cell line and found a mutation at position 273, Arg----His, with no normal allele present. The same mutation was also present in DNA from the tumour of origin. However immunohistochemical analysis of 129 human thyroid tumours using a panel of p53 antibodies was unequivocally negative. Southern blotting in 20 cases failed to demonstrate any deletion or rearrangement, and direct genomic sequencing of 20 carcinomas showed normal DNA sequence for exons 5, 7 and 8. Thus p53 abnormalities may not be important in human thyroid carcinogenesis, in contrast to colon, breast and lung. However, the FTC 133 cell line was only established after 132 unsuccessful attempts with other differentiated thyroid follicular tumours. Since this line and the corresponding tumour of origin have a p53 mutation, we propose that p53 mutation may confer on thyroid follicular tumour cells the ability to grow in culture. This has potential applications for the future development of thyroid carcinoma cell lines.     

Role of p53 and p38 MAP kinase in nitric oxide-induced G2/M arrest and apoptosis in the human lung carcinoma cells

The authors, Jui-I.Chao and Pao-Chen Kuo, found critical problemswith some of the data they reported in the above paper and wishto withdraw it from publication. The paper has been publishedonline     

Clinical value of alterations in p73 gene, related to p53 at 1p36, in human hepatocellular carcinoma

A novel gene, p73, encoding a protein with significant homology to p53 and showing functional similarities to p53, was identified at chromosome 1p36, at which tumor suppressor gene of hepatocellular carcinoma (HCC) is supposed to be. Involvement of p73 in hepatocarcinogenesis is controversial and clinical value of p73 alterations remains obscure. We investigated allelic status of p73 in 63 patients with HCC. Loss of heterozygosity (LOH) in p73 was analyzed by PCR-RFLP analysis. The results were compared with LOH on chromosome 1p surrounding p73 locus, mutations of p53 and p73, and clinicopathologic characteristics. LOH on p73 was observed in 33% of informative tumors. LOH in p73 was not always observed between the regions with LOH on chromosome 1p examined despite the significant association of LOH in p73 with LOH on chromosome 1p. No mutations were detected in p73. Tumors with LOH in p73 were more frequently detected in liver without cirrhosis than that with cirrhosis. There was no significant statistic association between the presence of LOH in p73 and six different clinicopathologic characteristics such as age, sex, histological type, T stage, tumor diameter, and virus status. Disease-free survival rates of the patients with LOH in p73 were significantly poorer than those without LOH in p73. Multivariate analysis indicated that presence of LOH in p73 was independent prognostic factor in patients with HCC. These findings suggested that p73 might play some role in tumor progression of HCC even though p73 should not be considered a candidate gene on chromosome 1p of HCC and does not function as a tumor suppressor gene like p53. Identifying the patients with LOH of p73 in tumors could be useful to predict early recurrence and to stratify the patients who need adjuvant therapy after operation.     

Wild-type p53 gene transfer resulted in cell cycle arrest, but not apoptosis of newly established human malignant fibrous histiocytoma cell line.

We established a human malignant fibrous histiocytoma (MFH) cell line, MFH-ToE, from a tumor originally developed in the right thigh of a 78-year-old woman. The original tumor histologically consisted of histiocytic, fibroblastic and giant cells. The tumor cells showed immunoreactivity for vimentin and alpha-1-antichymotrypsin, and were positive for acid phosphatase and non-specific esterase, being compatible with MFH. Although the histology of the heterotransplanted tumor into nude mice was similar to that of the primary MFH, the population of giant cells gradually decreased along with the culture passages. Cytogenetic analysis revealed a highly aneuploid nature with varying numbers of chromosomes from 71 to 140. Chromosome 17 showed monosomy and exon 6 to 8 of p53 gene was not amplified by PCR, implying absence of p53 function. Adenovirus vector-mediated wild-type p53 gene was successfully transfected into the MFH-ToE, which showed up-regulation of P53 and P21, as well as gradual up-regulation of Bcl-2 protein. The transfection resulted in cell cycle arrest, but not apoptosis of the MFH-ToE cells. These results revealed unique properties of the MFH-ToE, which might be useful in further studies analyzing pathological and biological characteristics of MFH.     

Expression of wild-type p53 in human A673 cells suppresses tumorigenicity but not growth rate

The p53 gene has been found to be mutated in many different kinds of human cancers. In a previous study, expression of exogenous wild-type p53 in human osteosarcoma cells by retrovirus-mediated gene transfer resulted in marked enlargement of cell size, reduced growth rate in culture and loss of tumorigenicity in nude mice. Here we examine the effects of expression of wild-type or mutated p53 on human peripheral neuroepithelioma (PNET) A673 cells; these cells contained apparently normal alleles of the p53 gene but did not express a detectable quantity of p53 protein. Various characteristics of the p53-expressing cells were examined including morphology, growth rate, soft-agar colony formation, and tumorigenicity in nude mice. In contrast to osteosarcoma Saos-2 cells, expression of wild-type or mutant p53 protein in A673 cells had no effect on morphology or growth characteristics. However, clones expressing wild-type p53 protein had reduced ability to form colonies in soft agar and tumors in nude mice. To substantiate the genotype of wild-type p53-expressing cells, the proviral p53-encoding DNA of one cell clone was amplified by the polymerase chain reaction and sequenced. We concluded that expression of a single allele of the wild-type p53 gene was sufficient to suppress PNET A673 tumorigenicity but had no detectable effect on growth rate in culture.