Colon Carcinoma Cell Line HCT116 Research Articles

Largazole and Analogues with Modified Metal-Binding Motifs Targeting Histone Deacetylases: Synthesis and Biological Evaluation

The histone deacetylase inhibitor largazole 1 was synthesized by a convergent approach that involved several efficient and high yielding single pot multistep protocols. Initial attempts using tert-butyl as thiol protecting group proved problematic, and synthesis was accomplished by switching to the trityl protecting group. This synthetic protocol provides a convenient approach to many new largazole analogues. Three side chain analogues with multiple heteroatoms for chelation with Zn(2+) were synthesized, and their biological activities were evaluated. They were less potent than largazole 1 in growth inhibition of HCT116 colon carcinoma cell line and in inducing increases in global H3 acetylation. Largazole 1 and the three side chain analogues had no effect on HDAC6, as indicated by the lack of increased acetylation of α-tubulin.

Characterization of dihydroartemisinin-resistant colon carcinoma HCT116/R cell line

Dihydroartemisinin (DHA) is an important artemisinin derivative and presents profound anti-tumor potential. A DHA-resistant cell line named HCT116/R derived from colon carcinoma cell line HCT116 was established in our previous study. Herein, we found that HCT116/R cells were much more resistant to DHA- or artesunate-induced proliferation inhibition and more tolerant to DHA-induced cell cycle arrest and apoptosis compared with those of the parent HCT116 cells. The protein levels of P-glycoprotein and MDR-associated protein 1 and the accumulation of doxorubicin in cells were similar in both cell lines. Moreover, HCT116/R cells were still sensitive to camptothecin- and doxorubicin-induced cell growth inhibition. To further explore the characterization of HCT116/R cell line, a proteomic study employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed. Eight different expressed proteins between the two cell lines were identified including some heat shock proteins, annexins, etc. This study not only indicates that exposure to DHA may not induce a tumor multi-drug-resistant phenotype but also affords new clues for the further investigation of the anti-cancer mechanisms of DHA and other artemisinin derivatives.

Bioactive constituents from Harpephyllum caffrum Bernh. and Rhus coriaria L

Background:The leaf ethanol extract of Harpephyllum caffrum Bernh. has evidenced medicinal value due to its hepatoprotective activity. It demonstrated inhibitory effects on test standard microbes approximated to 40% the potency of ofloxacin and fluconazole. The same extract evidenced in vitro cytotoxicity on human cell lines, liver carcinoma HEPG2, larynx carcinoma HEP2, and colon carcinoma HCT116 cell lines when compared to doxorubicin.Materials and Methods:Fractionation of the leaf ethanol extract led to the isolation of the polyphenols, ethyl gallate, and quercetin-3-O-rhamnoside, a hydrocarbon, hendecane, the fatty acid ester, methyl linoleate, and four triterpenoids, betulonic acid, 3-acetyl-methyl betulinate, lupenone and lupeol for the first time, in addition to the previously reported phenol acids and flavonoids, gallic acid, methyl gallate, quercetin, kaempferol, kaempferol-3-O-rhamnoside, kaempferol-3-O-galactoside, apigenin-7-O-glucoside, and quercetin-3-O-arabinoside.Results:The ethanol extract of the fruit of the genetically related Rhus coriaria L., known as sumac, afforded protocatechuic acid, isoquercitrin, and myricetin-3-O-α-L-rhamnoside from the fruits for the first time, in addition to the previously reported phenol acids and flavonoids, gallic acid, methyl gallate, kaempferol, and quercetin.Conclusion:The leaf ethanol extract of H. caffrum Bernh. exhibited variable anti-inflammatory, analgesic, and antipyretic activities, besides the hepatoprotective, in vitro cytotoxic and anti-microbial activities.

Novel Indolocarbazole Derivative 12-(α-L-arabinopyranosyl)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione Is a Preferred c-MycGuanine Quadruplex Ligand

The indolocarbazole derivative 12-(α-L-arabinopyranosyl)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (AIC) has demonstrated a high potency (at nanomolar to submicromolar concentrations) towards the NCI panel of human tumor cell lines and transplanted tumors. Intercalation into the DNA double helix has been identified as an important prerequisite for AIC cytotoxicity. In this study, we provide evidence for preferential binding to the G-quadruplex derived from the c-Myc oncogene promoter (Pu18 d(AG3TG4)2; G-c-Myc). The association constant for AIC:G-c-Myc complex was ~100 times and 10 times greater than the respective values for the complexes AIC:c-Myc duplex and AIC:telomeric d(TTAGGG)4 G-quadruplex. The concentrations at which AIC formed complexes with G-c-Myc were close to those that attenuated the steady-state level of the c-Myc mRNA in the human HCT116 colon carcinoma cell line. We suggest that preferential binding of AIC to G-c-Myc rather than to the c-Myc duplex might favor the quadruplex formation in the cells, thereby contributing to downregulation of the c-Myc expression by AIC.

Functional and Physical Interaction Between the Mismatch Repair and the FA-BRCA Pathways.

AbstractAbstract 3370 Introduction:Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, an increased risk for cancer and leukemia, and congenital abnormalities. Thirteen FA genes have been identified that when mutated result in hypersensitivity to DNA crosslinking agents. Therefore, components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). The monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI, is considered the hallmark of FA pathway activation. It has been reported that FANCJ interacts with the mismatch repair (MMR) complex MutLα and we have previously shown that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation of FANCD2 and FANCI. Interestingly, mismatch repair deficiency is also associated with leukemia in humans and a defect in hematopoietic repopulation in mouse models, suggesting a possible functional overlap between the FA-BRCA and MMR pathways. Methods:Cell lines used: HeLa, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agents mitomycin C (MMC), cisplatin (CDDP), or the alkylating agent temozolomide (TMZ). FANCD2 foci formation was assessed using immunofluorescence. Immunoprecipitation was used to demonstrate interactions between FANCD2, MSH2, and MLH1. Survival assays were performed by crystal violet staining and extraction. Chromosome breakage analysis was performed using metaphase spreads. FANCD2 RNAi flies, spel1-/- flies (MSH2-deficient), and double mutants were treated with diepoxybutane (DEB) and assessed for survival and mutagenicity. Mismatch binding EMSAs were performed using Cy5-labeled matched and mismatched 29-mers. Result:FANCD2 foci formation and chromatin loading is greatly diminished in MSH2-deficient cells, while cells lacking MLH1 show no effect, indicating a requirement for MSH2 in the activation of the FA-BRCA pathway and a possible downstream role for MLH1 in ICL repair. Human and mouse cells lacking MSH2 or human cells lacking MLH1 display increased sensitivity to mitomycin C and cisplatin as compared to their corrected counterparts as well as increased radial formation upon treatment with MMC. Studies in both human cell lines and Drosophila mutants indicate an epistatic relationship between FANCD2 and MSH2 with regards to survival, chromosome breakage, and mutagenicity after treatment with crosslinking agents. MSH2 is required for the interaction between FANCD2 and MLH1, but surprisingly, presence of MLH1 appears to enhance the interaction between FANCD2 and MSH2. Furthermore, the interaction between MSH2 and MLH1 after treatment with MMC is reduced in multiple FA cell lines. FA cell lines also display hypersensitivity to alkylating agents such as temozolomide. These results have led us to examine FA cell lines for a defect in mismatch repair through mismatch binding EMSAs and plasmid-based assays. Conclusion:These data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway. Conversely, FA proteins appear to be required for interactions between mismatch repair factors and may also be involved in the repair of DNA mismatches, suggesting significant crosstalk between the FA and MMR pathways. Understanding this complex interplay could lead to the development of new therapies for the treatment of patients both with FA and cancer. Disclosures:No relevant conflicts of interest to declare.

A partially degraded product of phytate suppresses the proliferation of HCT116 colorectal cancer cells

Phytate, myo-inositol hexaphosphate (IP6), has been shown to possess anti-carcinogenic qualities. IP6 appears to be partially digested in the digestive tract and degraded into inositol di, tri-, and tetra-phosphate (IP2, IP3, and IP4). The present study investigated whether IP3-rich phytate hydrolysate (IP3-RPH), a partially degraded product from phytate, plays a role in the suppression of cell proliferation, using an HCT116 colon carcinoma cell line. IP3-RPH suppresses cell proliferation as does IP6, and the suppression activity of IP3-RPH is higher than that of IP6. Suppression modes clearly differed between IP3-RPH and IP6. Our findings suggest that partially degraded IP6 products are responsible for the suppression of colon carcinogenesis by IP6.

Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression

Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G 1 phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21 Waf1/Cip1 and p27 Kip1; and knockdown of p27 kip1 with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.

Synthesis and cytotoxic potency of novel tris(1-alkylindol-3-yl)methylium salts: Role of N-alkyl substituents

Novel derivatives of tris(indol-3-yl)methane and tris(indol-3-yl)methylium salts with the alkyl substituents at the N-atoms of the indole rings were synthesized. An easy substitution of indole rings in trisindolylmethanes for other indoles under the action of acids is demonstrated, and the mechanism of substitution is discussed. To obtain trisindolylmethylium salts, the environmentally safe method of oxidation of trisindolylmethanes with air oxygen in acidic conditions was developed. Tris(1-alkylindol-3-yl)methanes and tris(1-alkylindol-3-yl)methylium salts represent three-bladed molecular propellers whose physico-chemical and biological properties strongly depend on the N-alkyl substituent. The cytotoxicity of novel compounds increased with the number of C atoms in the alkyl chains, with optimal number n=3–5 whereas the derivatives with longer side chains were less cytotoxic. The most potent novel compounds killed human tumor cells at nanomolar-to-submicromolar concentrations, being one order of magnitude more potent than the prototype antibiotic turbomycin A [tris(indol-3-yl)methylium salt]. Apoptosis in HCT116 colon carcinoma cell line induced by tris(1-pentyl-1H-indol-3-yl)methylium methanesulfonate was detectable at concentrations tolerable by normal blood lymphocytes. Thus, N-alkyl substituted tris(1-alkylindol-3-yl)methylium salts emerge as perspective anticancer drug candidates.

Action of SN 28049, a new DNA binding topoisomerase II-directed antitumour drug: comparison with doxorubicin and etoposide

We have examined the cellular action of SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide), a DNA binding drug with curative activity against the Colon 38 transplantable murine carcinoma, on human tumour cells. Its action has been compared with that of two topoisomerase II-targetted drugs, etoposide and doxorubicin. The NZM3 melanoma and HCT116 colon carcinoma cell lines, each expressing wild-type p53, were cultured and responses were compared by flow cytometry, electrophoresis, microscopy, and growth of tumour xenografts. Responses of NZM3 cells to all three drugs, as measured by histone H2AX γ-phosphorylation, induction of the p53 pathway and cell cycle arrest, were comparable and typical of those of topoisomerase II poisons. Xenografts of NZM3 cells responded to SN 28049 with a tumour growth delay of 16 days. In contrast, HCT116 cells had an attenuated DNA damage response to the drugs and SN 28049 had no in vivo activity, consistent with low topoisomerase II activity. However, SN 28049 inhibited HCT116 cell growth in vitro and activated the p53 pathway to induce a state with G(2)/M-phase DNA content, low mitotic index and a high proportion of binucleate cells. Treated cells expressed cyclin E and the senescence marker β-galactosidase but showed low expression of cyclin B and survivin. In comparison, etoposide caused little p53 expression or cycle arrest, and doxorubicin had an intermediate effect. The action of SN 28049 in NZM3 cells is typical of a topoisomerase II poison, but the low topoisomerase IIα activity of HCT116 cells allowed the detection of a second antiproliferative action of SN 28049 in which cells undergo post-mitotic cycle arrest and induction of p53.

Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription

The cytosine analogues 5-azacytidine and 5-aza-2'-deoxycytidine are currently the most advanced drugs for epigenetic cancer therapy. Both drugs function as DNA methyltransferase (DNMT) inhibitors and lead to the reactivation of epigenetically silenced tumour suppressor genes. However, not much is known about their target sequence specificity and their possible side effects on normally methylated sequences such as long interspersed nuclear element (LINE)-1 retroelements. It has been shown that demethylation and activation of the LINE-1 antisense promoter can drive the transcription of neighbouring sequences. In this study, we show that demethylation of the colon carcinoma cell line HCT116, either by treatment with DNMT inhibitors or by genetic disruption of the major DNMTs, induces the expression of an illegitimate fusion transcript between an intronic LINE-1 element and the proto-oncogene cMet (L1-cMet). Similar findings were also obtained with myeloid leukaemia cells, an established cellular model for the approved indication of azacytidine and decitabine. Interestingly, upregulation of L1-cMet transcription resulted in reduced cMet expression, which in turn led to decreased cMet receptor signalling. Our results thus provide an important paradigm for demethylation-dependent modulation of gene expression, even if the promoter of the corresponding gene is unmethylated.

Antiproliferative Pt(IV) complexes: synthesis, biological activity, and quantitative structure–activity relationship modeling

Several Pt(IV) complexes of the general formula [Pt(L)2(L')2(L'')2] [axial ligands L are Cl-, RCOO-, or OH-; equatorial ligands L' are two am(m)ine or one diamine; and equatorial ligands L'' are Cl- or glycolato] were rationally designed and synthesized in the attempt to develop a predictive quantitative structure-activity relationship (QSAR) model. Numerous theoretical molecular descriptors were used alongside physicochemical data (i.e., reduction peak potential, Ep, and partition coefficient, log Po/w) to obtain a validated QSAR between in vitro cytotoxicity (half maximal inhibitory concentrations, IC50, on A2780 ovarian and HCT116 colon carcinoma cell lines) and some features of Pt(IV) complexes. In the resulting best models, a lipophilic descriptor (log Po/w or the number of secondary sp3 carbon atoms) plus an electronic descriptor (Ep, the number of oxygen atoms, or the topological polar surface area expressed as the N,O polar contribution) is necessary for modeling, supporting the general finding that the biological behavior of Pt(IV) complexes can be rationalized on the basis of their cellular uptake, the Pt(IV)-->Pt(II) reduction, and the structure of the corresponding Pt(II) metabolites. Novel compounds were synthesized on the basis of their predicted cytotoxicity in the preliminary QSAR model, and were experimentally tested. A final QSAR model, based solely on theoretical molecular descriptors to ensure its general applicability, is proposed.

657 Protein kinase C and epidermal growth factor receptor signalling mediate growth stimulation by neurotensin in the colon carcinoma cell line HCT116

The recent discovery that the HMG box transcription factor XTCF-3 is involved in early axis specification in Xenopus laevis (Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J. Godsave, S., Korinek, V., Roose, J., Destrée, O., Clevers, H., 1996. XTcf-3 transcription factor mediates β-catenin-induced axis formation in Xenopus embryos. Cell 86, 391–399) led us to search for other members of the TCF/LEF family in this species. A newly identified HMG box factor was cloned with highest homology to human LEF-1, called XLEF-1. Unlike XTcf-3, XLef-1 is not expressed maternally, but its transcripts become detectable directly after the mid blastula transition (MBT). At later stages, both genes are expressed in the central nervous system (CNS), eyes, otic vesicles, head mesenchyme, neural crest and derivatives, branchial arches, developing heart, tailbud and limb buds. The expression pattern of Lef-1 during later stages of development is evolutionarily conserved.

Sodium 5,6‐benzylidene‐L‐ascorbate induces oxidative stress, autophagy, and growth arrest in human colon cancer HT‐29 cells

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT-29 cells with SAA and SBA, and found a significant exposure time-dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC(50) = 5 mM) than SBA (IC(50) = 10 mM). A short-term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase-dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21(+/+) and p21(-/-)), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co-incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N-acetyl-L-cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3-II were found in SBA-treated HT-29 cells; implicating that SBA induced AKT phosphorylation-autophagy and p21-growth arrest in colon cancer HT-29 cells through an NAC-inhibitable oxidative stress pathway.

Cryptosphaerolide, a Cytotoxic Mcl-1 Inhibitor from a Marine-Derived Ascomycete Related to the Genus Cryptosphaeria

Examination of the saline fermentation products from the marine-derived ascomycete fungal strain CNL-523 (Cryptosphaeria sp.) resulted in the isolation of cryptosphaerolide (1). The new compound is an ester-substituted sesquiterpenoid related to the eremophilane class. The structure of the new compound was assigned by spectroscopic and chemical methods. Cryptosphaerolide was found to be an inhibitor of the protein Mcl-1, a cancer drug target involved in apoptosis. It also showed significant cytotoxicity against an HCT-116 human colon carcinoma cell line, indicating that the compound may be of value in exploring the Mcl-1 pathway as a target for cancer chemotherapy.

β-Ionone Enhances TRAIL-Induced Apoptosis in Hepatocellular Carcinoma Cells through Sp1-Dependent Upregulation of DR5 and Downregulation of NF-κB Activity

beta-Ionone (ION), an end-ring analogue of beta-carotenoid, has been known to inhibit tumor cell growth and induce apoptosis in various types of cancer cells. Nevertheless, its apoptosis-related molecular mechanisms remain unclear. Here, we first investigated the molecular mechanisms by which ION sensitizes cancer cells to the therapeutic potential of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, treatment with subtoxic concentrations of ION and TRAIL effectively inhibited cell viability in the hepatocellular carcinoma cell line Hep3B and other cancer cell lines such as colon carcinoma cell line HCT116 and leukemia cell line U937. Combined treatment with ION and TRAIL was also more effective in inducing DR5 expression, caspase activities, and apoptosis than treatment with either agent alone. ION-mediated sensitization to TRAIL was efficiently reduced by treatment with a chimeric blocking antibody or small interfering RNA specific for DR5. Electrophoretic mobility shift assay and a chromatin immunoprecipitation assay confirmed that ION treatment upregulates the binding of transcription factor Sp1 to its putative site within the DR5 promoter region, suggesting that Sp1 is an ION-responsive transcription factor. In addition, ION significantly increased hepatocellular carcinoma cell sensitivity to TRAIL by abrogating TRAIL-induced NF-kappaB activation and decreasing the expression of antiapoptotic proteins such as XIAP and IAP-1/2. Taken together, these data suggest that ION is a useful agent for TRAIL-based cancer treatments. Mol Cancer Ther; 9(4); 833-43. (c)2010 AACR.

Gain of oncogenic function of p53 mutants regulates E-cadherin expression uncoupled from cell invasion in colon cancer cells

Mutations in the p53 tumour suppressor gene are associated clinically with tumour progression and metastasis. Downregulation of the E-cadherin cell-cell adhesion molecule is a key event for epithelial to mesenchymal transition (EMT) in tumour progression. Here, we show that wild-type p53 induced to adopt a mutant conformation, and hot-spot p53 mutants, which are both transcriptionally inactive, downregulate E-cadherin expression in the colon carcinoma cell line HCT116. Downregulation of E-cadherin occurred concomitantly with the upregulation of Slug and Zeb-1, transcriptional factors known to repress E-cadherin gene expression. In addition, knockdown of Slug and Zeb-1 expression diminished p53-mediated E-cadherin repression. Knocking down endogenous mutant p53 in MDA-MB-231 and SW620 cancer cell lines lacking E-cadherin protein restored the expression of E-cadherin. Complete loss of E-cadherin expression in HCT116 cells induced morphological alterations along with upregulation of vimentin, a mesenchymal marker. These changes characteristic of the EMT phenotype were, however, not sufficient to confer invasiveness in a three-dimensional matrix. Downregulation of E-cadherin by mutant p53 was not required to promote the invasive phenotype induced by inactivation of p53. These findings indicate that independent control of E-cadherin expression and cell motility could be essential molecular events in p53 mutant-induced invasive phenotypes.

A Role for Mismatch Repair Factors in the FA-BRCA Pathway.

Abstract 1095Poster Board I-117 IntroductionFanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). Central to this pathway is the monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI. Recent reports implicate mismatch repair factors in the repair of ICLs and have shown that FANCJ interacts with the MutLαa complex. Here we show that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation and chromatin localization of both FANCD2 and FANCI. MethodsCell lines used: HeLa, human lung carcinoma cell line H1299, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agent mitomycin C (MMC). Immunoprecipitation was used to demonstrate the interaction between FANCD2 and MSH2, MLH1, and MSH3. Survival assays were performed by crystal violet staining and extraction. Chromatin loading of FANCD2 and FANCI was determined by cellular fractionation and western blot. ResultsThrough chromatographic purification of FANCD2-containing protein complexes, we identified MSH2 and MLH1 as FANCD2-interacting proteins. Immunoprecipitation using HeLa cell extracts confirmed the interaction between FANCD2, MSH2, MSH3, and MLH1 in vivo. These interactions are all induced upon damage with a DNA crosslinking agent and MSH2 specifically interacts only with the monoubiquitylated form of FANCD2. Additionally, the FANCD2-MSH2 interaction requires ATR, but not ATM, BRCA1, MSH3, or ERCC1/XPF. Human cells lacking MSH2 display increased sensitivity to mitomycin C as compared to their corrected counterparts. FANCD2 and FANCI monoubiquitylation is also greatly diminished in these cells, while cells lacking MLH1 show no effect. Cellular fractionation of MSH2-deficient cells shows that FANCD2 and FANCI are not efficiently loaded onto chromatin after treatment with DNA-damaging agents, while MLH1-deficient cells again show no effect. Interestingly, while knockdown of either MSH2 or FANCD2 in H1299 cells results in increased sensitivity to MMC, double knockdown of both proteins corrects this sensitivity on par with controls. oConclusionsThese data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway, likely through recognition of the DNA lesion. Understanding this role could lead to the development of new therapies for the treatment of patients both with FA and cancer. DisclosuresNo relevant conflicts of interest to declare.

Inhibitory effect of low molecular weight heparin on the secretion of vascular endothelial growth factor by tumor cells in vitro

To investigate whether low molecular weight heparin (LMWH) may suppress the expression and secretion of vascular endothelial growth factor (VEGF) from tumor cells in vitro and inhibit the VEGF-induced proliferation of human tumor vascular endothelial cells. Human lung cancer cell line A549, human liver cancer cell line HepG2, human colon carcinoma cell lines HCT116 and HCT8 were used in this study. The expression levels of VEGF and TNF-alpha (tumor necrosis factor-alpha) in the tumor cells with or without pretreatment of LMWH/heparin were measured by standard sandwich ELISA technique. The VEGF mRNA level of HepG2 cells cultured with or without LMWH/heparin was determined by RT-PCR and real time PCR. Human umbilical vein endothelial cells (HUVEC) were cultured in tissue culture medium (TCM) with or without LMWH/heparin for 3 days. Then non-radioactive cell proliferation assay (MTS) kit and cell cycle assay by flow cytometry were performed to measure the proliferation of HUVEC. The VEGF levels in the control, LMWH, and heparin groups of the pulmonary adenocarcinoma cell line A549 were (1045.89 +/- 165.30) pg/ml, (782.45 +/- 67.17) pg/ml and (916.54 +/- 71.25) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups of the colon adenocarcinoma cell line HCT116 were (955.76 +/- 51.14) pg/ml, (822.89 +/- 142.39) pg/ml and (951.77 +/- 188.22) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the colon adenocarcinoma cell line HCT8 were (1290.62 +/- 41.23) pg/ml, (1063.34 +/- 63.82) pg/ml and (1257.14 +/- 11.40) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the liver cancer cell line HepG2 were (1083.00 +/- 134.35) pg/ml, (758.00 +/- 84.85) pg/ml and (874.00 +/- 22.62) pg/ml, respectively. The VEGF expression levels in the above mentioned cell lines cultured in TCM were significantly reduced in the LMWH-treated groups compared with that of the control group (P < 0.05). But the level of TNF-alpha in TCM-cultured cells was unaffected by LMWH. The VEGF mRNA was reduced in the LMWH-treated HepG2 cell line. Moreover, TCM exhibited stimulating effect on proliferation of HUVEC and the effect was significantly impaired by LMWH treatment. Flow cytometric analysis revealed that LMWH treatment arrested HUVECs at the G1 phase of cell cycle. LMWH can suppress the expression and secretion of VEGF by tumor cell lines and therefore have a potential inhibiting effect on angiogenesis induced by VEGF.

The role of p53 in the cellular toxicity by active trans-platinum complexes containing isopropylamine and hydroxymethylpyridine

Despite some initial research that reported a lack of activity of trans geometry, complexes with general formula trans-[PtCl 2(L)(L′)] exhibit an important cytotoxic activity in cisplatin-sensitive and resistant cell lines. Based on the proposed mechanism of action for the trans-platinum compounds, they might form DNA adducts initiating a DNA-damage response and ultimately ending in the activation of the p53 protein. In the present work, we have studied the biochemical properties of the trans-[PtCl 2(isopropylamine)(L)] complexes (where L is 3- or 4-(hydroxymethyl)-pyridine) against several cell lines and the relationship between cytotoxicity and the protein p53. Both complexes showed different antitumoral properties depending on the presence or absence of protein p53 in isogenic colon carcinoma HCT116 cell lines. Cell cycle studies with the complexes in these cell lines were performed to investigate their antitumoral activity. Apoptosis was observed to be launched from G1 or G2/M accumulations. Confocal microscopy showed the different behaviour of isogenic tumoral cell lines treated with the trans-platinum complexes. Our data suggest that small differences in the carrier ligands could play an important role in the overall biological effects. The body of the research regarding structure–activity relationships such as the different position of groups in the carrier ligands will provide new rational basis for the design of new platinum antitumor drugs.

Bingchamides A and B, two novel cyclic pentapeptides from the Streptomyces bingchenggensis: fermentation, isolation, structure elucidation and biological properties

Two novel cyclic pentapeptides, bingchamides A (1) and B (2), have been isolated from the organic extracts of the mycelium of Streptomyces bingchenggensis. The structures of 1 and 2 were elucidated on the basis of extensive 1D and 2D NMR, as well as HRESI-MS, electrospray ionization-MS, UV and IR spectroscopic data analysis. Bingchamides A (1) and B (2) exhibited in vitro cytotoxicity toward human colon carcinoma cell line HCT-116 with the IC(50) values of 14.1 and 18.0 microg ml(-1), respectively. The bingchamides A (1) and B (2) scaffolds are probably promising structures for the development of new antitumor agents.