海绵动物抗肿瘤活性物质研究进展

海洋生物是抗肿瘤活性物质的重要来源。海绵动物是海洋中除珊瑚以外的第2大生物资源。近十几年来,已从不同的海绵体内分离鉴定了许多结构新颖的高活性抗肿瘤物质,显示出诱人的研究开发前景。现综述正在临床试验阶段的具有抗肿瘤活性的海绵动物提取物及其合成衍生物,以及其来源种属、化学成分及其药理作用的研究进展。     

海绵动物抗肿瘤活性物质研究进展

海洋生物是抗肿瘤活性物质的重要来源。海绵动物是海洋中除珊瑚以外的第2大生物资源。近十几年来,已从不同的海绵体内分离鉴定了许多结构新颖的高活性抗肿瘤物质,显示出诱人的研究开发前景。现综述正在临床试验阶段的具有抗肿瘤活性的海绵动物提取物及其合成衍生物,以及其来源种属、化学成分及其药理作用的研究进展。     

Cetuximab potentiates oxaliplatin cytotoxic effect through a defect in NER and DNA replication initiation

Preclinical studies have demonstrated that the chemotherapeutic action of oxaliplatin, a third generation platinum derivative, is improved when combined with cetuximab, a monoclonal antibody inhibitor of epidermal growth factor receptors. To explore the mechanism of this synergistic benefit, we used HCT-8 and HCT-116, two human colon cancer cell lines, respectively, responsive and non-responsive to the oxaliplatin/cetuximab combination. We examined the effect of drug exposure on glutathione-S-transferase-mediated oxaliplatin detoxification, DNA-platinum adducts formation, cell cycle distribution, apoptosis, and the expression of multiple targets involved in DNA replication, recombination, and repair. The major changes we found in HCT-8 were a stimulation of oxaliplatin-DNA adduct formation associated with reduced expression of the key enzyme (excision repair cross complementation group1: ERCC1) in the key repair process of oxaliplatin-DNA platinum adduct, the nucleotide excision repair (NER), both at the mRNA and protein levels. We also observed a reduced expression of factors involved in DNA replication initiation, which correlates with an enrichment of cells in the G1 phase of the cell cycle as well as an acceleration of apoptosis. None of these changes occurred in the non-responsive HCT-116 cell that we used as a negative control. These findings support the fact that cetuximab potentiates the oxaliplatin-mediated cytotoxic effect as the result of inhibition of NER and also DNA replication initiation.     

Gene expression rather than the initiation of DNA replication is the principal target of lethal u.v.-induced damage in a regulatory region of SV40 DNA

The survival of transfected simian virus (SV) 40 DNA is acutelysensitive to damage in a 302-bp regulatory region that governsviral gene expression and the initiation of viral DNA replication.We investigated whether the lethal effect of damage in thisregion is due to the disruption of gene expression or to theinhibition of DNA replication by comparing the survival of damagedviral DNA in CV-1 and cos-1 African green monkey cells. Viralearly sequences integrated into the genomic DNA of cos-1 cellscomplement the growth of virus with defective early genes andwere therefore expected to reverse viral sensitivity to lesionsthat interfere with early gene expression. Our results indicatethat viral sensitivity to damage in the regulatory region isalmost completely abolished in cos-1 cells. This finding identifiesgene expression rather than the initiation of DNA replicationas the major target for lethal damage in that portion of theSV40 genome. Sensitivity to damage in the viral late gene regionis the same in CV-1 and cos-1 cells, indicating that cos-1 cellsare not merely more proficient in host-cell reactivation. Ourresults allow us to partition the overall lethal effect of DNAdamage into sectors, and to assign each sector to the disruptionof a particular genetic function.     

Diagnosis of cancer at the DNA level

Recent recombinant DNA techniques have made possible the production of gene probes and the search for genetic damage in neoplastic cells, and now occupy one of the central place in cancer research. More recently, detection of immunoglobulin and T cell receptor gene rearrangements has been shown to be a powerful procedure for identifying monoclonality and the cellular lineage of lymphoid cells even when conventional studies give an ambiguous diagnosis. Such genetic markers are not only useful for differential diagnosis and classification, but serves also as a sensitive unique clonal marker to detect early cancer relapse. In a similar manner, chromosomal translocations associated with specific disease types can be detected with DNA probes in southern blot analysis without the use of conventional cytogenetics. These methods have wider application and will play an increasing role in the clinical use in the near future.     

Apoptotic activity of ursolic acid may correlate with the inhibition of initiation of DNA replication

Ursolic acid (UA), a pentacyclic triterpene acid, has been reported to exhibit anti-tumor activity. In this study, we investigated the pro-apoptotic effect of UA on HepG2 human hepatoblastoma cells. Treatment with UA decreased the viability of HepG2 cells in a concentration- and time-dependent manner. Furthermore, 30 microM of UA induced DNA fragmentation and subdiploid cells and enhanced the release of cytochrome c and the activation of caspase-3. These results suggest that UA induces cell death through apoptosis, which may be mediated by cytochrome c-dependent caspase-3 activation. In addition, cell-cycle analysis revealed that UA-treated cells were arrested predominantly in the G(0) and G(1) phases with a concomitant decrease in the cell population of S phase. Moreover, expression of p21(WAF1), a cell-cycle regulator, was increased by UA, indicating that p21(WAF1) might mediate UA-induced cell-cycle arrest. However, UA markedly inhibited SV40 DNA replication in the initiation stage in vitro and significantly reduced the DNA cleaving of topoisomerase I and the ssDNA binding activity of replication protein A. These results indicate that the inhibition of DNA replication by UA may result from blockade of the establishment of the replication fork during initiation stage, consequently contributing to UA-induced cell-cycle arrest. Taken together, we suggest that UA-induced cell-cycle arrest may be mediated by inhibition of DNA replication and the increase of p21(WAF1) expression, which induces the release of cytochrome c and the activation of caspase-3, leading to apoptosis of HepG2 cells.     

Cytotoxicity of p-tyrosol and its derivatives may correlate with the inhibition of DNA replication initiation

p-Tyrosol is a phenolic compound present in different dietary sources that can exert mild antioxidant properties based on in vitro and in vivo studies. In our study, two p-tyrosol derivatives (p-tyrosyl gallate and p-tyrosyl acetate) were synthesized and compared together with p-tyrosol and gallic acid for their cytotoxic activities on human cancer cells. p-Tyrosyl gallate had the most potent cytotoxicity and the major cytotoxic mechanism of its action was studied. We found that in HeLa cells, p-tyrosyl gallate can effectively induce cell cycle arrest during S phase and inhibited in vitro simian virus (SV40 DNA) replication. In addition, p-tyrosyl gallate can inhibit three important functional replication proteins (topoisomerase I, RPA and pol alpha-primase), especially pol alpha-primase. These results suggest that p-tyrosyl gallate-induced cell cycle arrest during S phase correlates with the inhibition of DNA replication. Pol alpha-primase may be the main target molecule. Taken together, we suggest that p-tyrosyl gallate is a strong anticancer drug candidate that warrants further investigation.     

Concentration dependent alterations of DNA replication initiation and elongation by benzo[a]pyrene diol epoxide

Vero cells treated with various concentrations of (±)7ß,8dihydroxy-9, 10-epoxy, 7,8,9,10-dihydrobenzo[a]pyrene (BP-diolepoxide I) exhibited dose-dependent inhibition in both the rateof DNA synthesis and in the size of nascent DNA. The maximuminhibition was seen 2–3 h after addition of BP-diol epoxideI. A recovery in both the rate of synthesis and size of nascentDNA was observed 5–10 h after treatment. The pH step alkalineelution assay which separates different nascent DNA replicationintermediates was used to investigate whether the inhibitionand recovery noted above could be accounted for by alterationsin DNA replication initiation (DNA synthesis within a replicon)or elongation (rejoining of replicons). At lowest dose studied(0.166 µM BPdiol epoxide I) a reversible inhibition inDNA initiation was observed. At the higher dose levels (0.66µM and 1.66 µM BPdiol epoxide I) inhibition of bothDNA initiation and elongation were observed and inhibition ofelongation predominated. The inhibition in elongation was detectedby an increase in the relative amount of low molecular weightnascent DNA associated with DNA synthesis within a repliconand a relative decrease in the higher molecular weight elongatedDNA. A reversal in the inhibition of both initiation and elongationwas noted.     

Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation

Many severely hypoxic cells fail to initiate DNA replication, but the mechanism underlying this observation is unknown. Specifically, although the ataxia-telangiectasia-rad3 related (ATR) kinase has been shown to be activated in hypoxic cells, several studies have not been able to document down-stream consequences of ATR activation in these cells. By clearly defining the DNA replication initiation checkpoint in hypoxic cells, we now demonstrate that ATR is responsible for activating this checkpoint. We show that the hypoxic activation of ATR leads to the phosphorylation-dependent degradation of the cdc25a phosphatase. Downregulation of cdc25a protein by ATR in hypoxic cells decreases CDK2 phosphorylation and activity, which results in the degradation of cdc6 by APC/C(Cdh1). These events do not occur in hypoxic cells when ATR is depleted, and the initiation of DNA replication is maintained. We therefore present a novel mechanism of cdc6 regulation in which ATR can have a central role in inhibiting the initiation of DNA replication by the regulation of cdc6 by APC/C(Cdh1). This model provides insight into the biology and therapy of hypoxic tumors.     

Antineoplastic agents 470. Absolute configuration of the marine sponge bromopyrrole agelastatin A

Two bromopyrrole marine alkaloids were isolated from the Mexican sponge, Agelas sp.: hymenidin (1) and agelastatin A (2). The structures were elucidated by analysis of their spectroscopic data and found to correspond to those in the literature. The absolute configuration of agelastatin A (2) was elucidated by single-crystal X-ray diffraction methods. Agelastatin A (2) exhibited strong activity against a panel of human cancer cell lines as well as human umbilical vein endothelial cells.     

The possible roles for polyamines in the initiation process of SV40 DNA replication in vitro

The polyamines are aliphatic cations which are present in millimolar concentrations in all mammalian cells, and are required for optimal growth of almost all cell types. In this study, the roles of polyamines in DNA replication in vitro and the mechanism by which polyamines affected DNA replication were examined using simian virus 40 DNA replication system in vitro. We found that polyamines inhibited DNA replication, but it is not clear at which stage this occurs. Spermidine inhibited the DNA cleavage by topoisomerase I at 8.0 mM, but stimulated its activity at 1.0 mM. Spermine also inhibited its activity at 4.0 mM, but stimulated at 1.0 mM. The ssDNA binding activity of replication protein A was slightly affected by polyamines. Polyamines, especially spermine, also significantly reduced polymerase alpha-primase activity at 133 microM. Taken together, we suggest that the major inhibition of SV40 DNA replication may be due to the inhibition of pol alpha-primase activity, and possible roles for polyamines in the initiation process are discussed.     

Inhibition of DNA replication by tirapazamine.

Tirapazamine (TPZ) is a hypoxia-selective cytotoxin that is currently being examined in Phase II and III clinical trials in combination with radiotherapy and cisplatin-based chemotherapy. Reductases convert TPZ to a cytotoxic radical that produces DNA damage under hypoxic conditions. Because one or more of the enzymes responsible for the bioactivation of TPZ is/are thought to be at or near the nuclear matrix, we hypothesized that TPZ may have a major affect on DNA replication, a process that is known to occur predominantly at the nuclear matrix. To assess the effect of TPZ on DNA replication, we measured the incorporation of radioactive thymidine into DNA of HCT116 human colon cancer cells and HeLa cells. We show that incorporation of radioactive thymidine is dramatically inhibited in cells that are pretreated with TPZ under hypoxic conditions. TPZ-induced inhibition of DNA synthesis was much greater than that produced by more toxic doses of ionizing radiation. We used the SV40-based in vitro DNA replication assay to study the mechanism of inhibition of DNA synthesis in cells treated with TPZ. Using this assay, we show that extracts prepared from cells treated with TPZ under hypoxic conditions had only 25-50% of the DNA replication activity measured in control cells. This reduction in DNA replication activity was associated with a reduction in levels of replication protein A (RPA) in cytoplasmic extracts used for the in vitro DNA replication assay and could be overcome by addition of recombinant human RPA. Furthermore, we show by indirect immunofluorescence that TPZ leads to a localization of the p34 subunit of RPA (RPA2) to small subnuclear foci. These results show that TPZ dramatically inhibits DNA replication and that the mechanism of inhibition, at least in part, involves changes in RPA that alter its cellular localization.     

Inhibition of initiation of simian virus 40 DNA replication in vitro by the ursodeoxycholic acid and its derivatives

We induced tolerance to hexadecylphosphocholine (HePC) in the human epidermoid tumor cell line, KB. After 70 weeks of adaptation, the IC50 of HePC in the resistant cells KBr was 32-fold higher than in parental KB cells, and they were 30-fold more resistant to another ether lipid analogue, ET-18-OCH3. The KBr cells also showed cross-resistance to vincristine and colchicine while remaining sensitive to other chemotherapy agents. RT-PCR assays showed that expression of the multidrug resistance gene (MDR1) was positive in KBr cells, whereas the expression of GST-pi (glutathione S-transferase pi) and MRP (multidrug resistance protein) was undetectable in KBr cells. Both an immunocytochemistry test and Western blot analysis indicated that the expression of bcl-2 in KBr cells was strongly positive, while it was only mildly expressed in KB cells. Verapamil could not reverse the resistance of KBr to HePC although it is a well-known reversing agent against MDR1. Our results suggest that bcl-2 instead of MDR1 plays a major role in the resistance of KBr cells.     

Chromatin remodelling and DNA replication: from nucleosomes to loop domains.

Organization of DNA into chromatin is likely to participate in the control of the timing and selection of DNA replication origins. Reorganization of the chromatin is carried out by chromatin remodelling machines, which may affect the choice of replication origins and efficiency of replication. Replication itself causes a profound rearrangement in the chromatin structure, from nucleosomes to DNA loop domains, allowing to retain or switch an epigenetic state. The present review considers the effects of chromatin remodelling on replication and vice versa.     

Phenotypic heterogeneity at the DNA level in childhood leukemia with a mediastinal mass

Although phenotypic heterogeneity of childhood T-cell acute lymphocytic leukemia (T-ALL) which bear receptors for sheep red blood cells (E-rosettes) and/or T-cell-associated antigens has been reported, there are certain clinical features which are shared by most patients. A mediastinal mass is one of the most characteristic presentations in this particular disorder. This report describes four children with ALL, who presented with a mediastinal mass. Three patients were E-rosette-negative and one was E-rosette-positive. Individual surface phenotypes, defined by a panel of monoclonal antibodies, were quite different. Since Ig gene organization is an essential property of cells of B-lineage, it was surprising to find that analysis of genomic DNA revealed immunoglobulin (Ig) gene rearrangements in two of them. These findings suggest that there is significant heterogeneity even among those leukemias associated with a mediastinal mass, and that a mediastinal mass may not clearly indicate origin from cells of T-lineage. This heterogeneity may reflect differences in leukemogenesis and may have prognostic and therapeutic implications.     

Intravesical chemotherapy of high-grade bladder cancer with HTI-286, a synthetic analogue of the marine sponge product hemiasterlin.

PURPOSE: HTI-286 is a fully synthetic analogue of the natural tripeptide hemiasterlin that inhibits tubulin polymerization and has strong cytotoxic potential. In this study, we evaluate the inhibitory effects of HTI-286 on human bladder cancer growth, both in vitro and as an intravesical agent in an orthotopic murine model. EXPERIMENTAL DESIGN: Various bladder cancer cell lines were treated with HTI-286 and mitomycin C (MMC) in vitro. Human KU-7 bladder tumor cells that stably express firefly luciferase were inoculated in female nude mice by intravesical instillation and quantified using bioluminescence imaging. Mice with established KU-7-luc tumors were given HTI-286 or MMC intravesically twice a week for 2 h. Pharmacokinetic data was obtained using high-performance liquid chromatography-mass spectrometry analyses. RESULTS: In vitro, HTI-286 was a potent inhibitor of proliferation in all tested cell lines and induced marked increases in apoptosis of KU-7-luc cells even after brief exposure. In vivo, HTI-286 significantly delayed cancer growth of bladder tumors in a dose-dependent fashion. HTI-286, at a concentration of 0.2 mg/mL, had comparable strong cytotoxicity as 2.0 mg/mL of MMC. The estimated systemic bioavailability of intravesically given HTI-286 was 1.5% to 2.1% of the initial dose. CONCLUSIONS: Intravesical HTI-286 instillation therapy showed promising antitumor activity and minimal toxicity in an orthotopic mouse model of high-grade bladder cancer. These findings provide preclinical proof-of-principle for HTI-286 as an intravesical therapy for nonmuscle-invasive bladder cancer and warrant further evaluation of efficacy and safety in early-phase clinical trials.     

Synergistic effects on the initiation of rat liver tumors by trans-4-acetylaminostilbene and 2-acetylaminofluorene, studied at the level of DNA adduct formation

Both trans-4-acetylaminostilbene (AM) and 2-acetylaminofluorene(AAF) exert tumor-initiating activity in rat liver when administeredin the initiation phase of an initiationpromotion experiment.The effects are more than additive when the compounds are sequentiallycombined in the initiation phase of such an experiment, andthis synergism is more pronaunced when AAS is given first, followedby AAF, than vice versa. In order to determine the role of targetDNA dose, [³H]AAS and [¹⁴C]AAF were admiaistered to female Wistarrats adhering to the protocol of the initiation phase of theabove-mentioned experiment and the following parameters measuredat the end of this phase: total radioactivity m tissues, bindingto DNA, RNA and proteins in liver, adduct pattern in liver DNAand RNA. In neither combination were these parameters significantlydifferent from those in the appropriate controls in which onlyone of the compounds was administered. This result indicatesthat combining the substances did not alter the pharmacokineticsof the individual compounds and that the target dose is additive.This suggests that effects unrelated to DNA binding, possiblypromding effects, may cause the more than additive generationof preneoplastic lesions in rat liver.