Established Human Cell Lines Research Articles

Sodium caprate as an enhancer of macromolecule permeation across tricellular tight junctions of intestinal cells

Sodium caprate is a promising candidate for inducing drug absorption enhancement. The mechanism of that uptake-enhancing effect is not fully understood so far. We investigated how caprate acts in an established human intestinal cell line, HT-29/B6, on the transient opening of transcellular (across the cell membranes) and paracellular (across the tight junction) pathways. Sodium caprate (10 mm) caused a rapid and reversible decrease of transepithelial resistance which is based, as measured by two-path impedance spectroscopy, exclusively on resistance changes of the paracellular pathway. Measurements of paracellular marker fluxes revealed an increased permeability for fluorescein (330 Da) and FITC-dextran (4 and 10 kDa), indicating an opening of the paracellular barrier. Confocal microscopy revealed a marked reduction of tricellulin in tricellular tight junctions and of claudin-5 in bicellular tight junctions. This was not due to altered protein expression, as occludin, claudins or tricellulin were not significantly changed in Western blots. Visualization of the translocation site of the cell membrane-impermeable marker molecule sulpho-NHS-SS-biotin (607 Da) indicated the tricellular tight junction to be the predominant pathway. We suggest that caprate's known enhancing effect on intestinal drug uptake is based on increased permeability in tricellular cell contacts, mediated by reversible removal of tricellulin from the tricellular tight junction.

Aurora kinase inhibitor VE 465 synergistically enhances cytotoxicity of carboplatin in ovarian cancer cells through induction of apoptosis and downregulation of histone 3

Aurora kinases are essential for regulation of chromosome segregation and cytokinesis during mitosis and play a role in growth and progression of human tumors, including ovarian cancer. Aurora A and Aurora B are frequently overexpressed in high-grade and low-grade ovarian cancers. Targeting Aurora kinases has great potential for improving the efficacy of chemotherapies of ovarian cancer. In this study, we investigated whether the Aurora kinase inhibitor, VE 465, can enhance the anti-tumor activity of carboplatin in human ovarian cancer cells. The antitumor activity of VE 465 was tested by MTT proliferative assay in multiple established human epithelial ovarian cancer cell lines of varying p53 status. VE 465 and carboplatin had a synergistic effect on cell viability in both platinum-sensitive and -resistant ovarian cancers. The growth-inhibitory effect was accompanied by reduction in expression of histone 3 and an increase in apoptosis. We conclude that VE 465 enhances the efficacy of carboplatin agents in ovarian carcinoma.

Three-dimensional Invasion of Human Glioblastoma Cells Remains Unchanged by X-ray and Carbon Ion Irradiation In Vitro

Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks (γH2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg, β1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the β1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.

CD133+, CD166+CD44+, and CD24+CD44+ Phenotypes Fail to Reliably Identify Cell Populations with Cancer Stem Cell Functional Features in Established Human Colorectal Cancer Cell Lines

Increasing evidence that cancers originate from small populations of so-called cancer stem cells (CSCs), capable of surviving conventional chemotherapies and regenerating the original tumor, urges the development of novel CSC-targeted treatments. Screening of new anticancer compounds is conventionally conducted on established tumor cell lines, providing sufficient material for high-throughput studies. Whether tumor cell lines might comprise CSC populations resembling those of primary tumors, however, remains highly debated. We have analyzed the expression of defined phenotypic profiles, including CD133+, CD166+CD44+, and CD24+CD44+, reported as CSC-specific in human primary colorectal cancer (CRC), on a panel of 10 established CRC cell lines and evaluated their correlation with CSC properties. None of the putative CSC phenotypes consistently correlated with stem cell-like features, including spheroid formation ability, clonogenicity, aldehyde dehydrogenase-1 activity, and side population phenotype. Importantly, CRC cells expressing putative CSC markers did not exhibit increased survival when treated with chemotherapeutic drugs in vitro or display higher tumorigenicity in vivo. Thus, the expression of CD133 or the coexpression of CD166/CD44 or CD24/CD44 did not appear to reliably identify CSC populations in established CRC cell lines. Our findings question the suitability of cell lines for the screening of CSC-specific therapies and underline the urgency of developing novel platforms for anticancer drug discovery.

Mouse Models as a Translational Platform for the Development of New Therapeutic Agents in Multiple Myeloma

Mouse models of multiple myeloma (MM) are basic tools for translational research and play a fundamental role in the development of new therapeutics against plasma cell malignancies. All available models, including transplantable murine tumors in syngenic mice, xenografts of established human cell lines in immunocompromised mice and transgenic models that mirror specific steps of MM pathogenesis, have demonstrated some weaknesses in predicting clinical results, particularly for new drugs targeting the human bone marrow microenvironment (huBMM). The recent interest to models recapitulating the in vivo growth of primary MM cells in a human (SCID-hu) or humanized (SCID-synth-hu) host recipient has provided powerful platforms for the investigation of new compounds targeting MM and/or its huBMM. Here, we review and discuss strengths and weaknesses of the key in vivo models that are currently utilized in the MM preclinical investigation.

1008 Characterization of Newly Established P-glycoprotein Over-expressing Multi-drug Resistant Human Cancer Cell Lines

1008 Characterization of Newly Established P-glycoprotein Over-expressing Multi-drug Resistant Human Cancer Cell Lines

Examination of the Cytotoxic and Embryotoxic Potential and Underlying Mechanisms of Next-Generation Synthetic Trioxolane and Tetraoxane Antimalarials

Semisynthetic artemisinin-based therapies are the first-line treatment for P. falciparum malaria, but next-generation synthetic drug candidates are urgently required to improve availability and respond to the emergence of artemisinin-resistant parasites. Artemisinins are embryotoxic in animal models and induce apoptosis in sensitive mammalian cells. Understanding the cytotoxic propensities of antimalarial drug candidates is crucial to their successful development and utilization. Here, we demonstrate that, similarly to the model artemisinin artesunate (ARS), a synthetic tetraoxane drug candidate (RKA182) and a trioxolane equivalent (FBEG100) induce embryotoxicity and depletion of primitive erythroblasts in a rodent model. We also show that RKA182, FBEG100 and ARS are cytotoxic toward a panel of established and primary human cell lines, with caspase-dependent apoptosis and caspase-independent necrosis underlying the induction of cell death. Although the toxic effects of RKA182 and FBEG100 proceed more rapidly and are relatively less cell-selective than that of ARS, all three compounds are shown to be dependent upon heme, iron and oxidative stress for their ability to induce cell death. However, in contrast to previously studied artemisinins, the toxicity of RKA182 and FBEG100 is shown to be independent of general chemical decomposition. Although tetraoxanes and trioxolanes have shown promise as next-generation antimalarials, the data described here indicate that adverse effects associated with artemisinins, including embryotoxicity, cannot be ruled out with these novel compounds, and a full understanding of their toxicological actions will be central to the continuing design and development of safe and effective drug candidates which could prove important in the fight against malaria.

Cytotoxicity and apoptosis-inducing effect of steroidal saponins from Dioscorea zingiberensis Wright against cancer cells

Steroidal saponins from Dioscorea zingiberensis Wright (DZW) have shown cytotoxic activity in cancer cells. In this study, we isolated and identified seven steroidal saponins from the rhizomes of DZW: diosgenin, trillin, diosgenin diglucoside, deltonin, zingiberensis saponin (ZS), protodeltonin and parvifloside. Our results showed that these seven compounds inhibited the proliferation of a panel of established human and murine cancer cell lines in vitro. ZS had more cytotoxic effect than the other saponins, even close to doxorubicin on the murine colon carcinoma cell line C26. The proliferation inhibitory effect of ZS was associated with its apoptosis-inducing effect by activation of caspase-3 and caspase-9 and specific proteolytic cleavage of poly (ADP-ribose) polymerase. Exposure of C26 to ZS also resulted in Bax upregulation and Bcl-2 downregulation. In conclusion, the findings of this study demonstrated that ZS is an effective natural agent for cancer therapy, which may be mediated, in part, by induction of apoptosis, and DZW’s potential as an anticancer agent is worth being further investigated.

Abstract 277: The small molecule tolfenamic acid inhibits neuroblastoma cell growth through inducing apoptosis and causing cell cycle arrest potentially targeting Sp1 and survivin

Abstract Neuroblastoma is an embryonal tumor of the peripheral sympathetic nervous system found in infants and children. Despite intensive therapy, the mortality rate of this malignancy remains more than 50%. The side effects of standard therapy can cause acute and long term damage and toxicity in various biological functions and can lead to the development of secondary tumors. Therefore there is an urgent need in identifying novel strategies in neuroblastoma that improve out come with less toxicity. Specificity proteins (Sp) regulate several important genes associated with neuroblastoma including MYCN and TrkA. Our aim was to exploit the strategy of targeting Sp proteins using TA in the treatment of neuroblastoma. In this study, we examined the anti-proliferative activity of TA using 7 neuroblastoma cell lines and evaluated the ability of TA to induce apoptosis, and cell cycle arrest using selected cell lines. Apoptosis in mammalian cells primarily takes place through extrinsic and intrinsic pathways. Specificity protein (Sp) transcription factors regulate several genes involved in cell proliferation, survival and angiogenesis. Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to induce the degradation of Sp proteins and may serve as a novel anti-cancer agent. The objective of this investigation was to examine the anti-cancer activity of TA using established human neuroblastoma cell lines. We tested the anti-proliferative effect of TA using 7 human neuroblastoma cell lines. Cells were treated with TA (0/25/50/100μM) and cell viability was measured at 24, 48, and 72 h post-treatment. Selected neuroblastoma cell lines, LA1 55n and SH-SY5Y were treated with 50 μM TA for 24 and 48 h and tested for cell apoptosis using Annexin-V staining. Caspase activity was measured with caspase 3/7 Glo kit. Cell lysates were prepared and the expression of Sp1, survivin and c-PARP were evaluated through Western blot analysis. TA significantly inhibited the growth of neuroblastoma cells in a dose/time-dependent manner and significantly decreased Sp1 and survivin expression. Apart from cell cycle (G0/G1) arrest, TA caused significant increase in the apoptotic cell population, caspase 3/7 activity and c-PARP expression. These results show that TA effectively inhibits neuroblastoma cell growth potentially through suppressing mitosis, Sp1 and survivin expression, and inducing apoptosis. These results represent a novel strategy for using small molecules for the treatment of neuroblastoma.(Financial assistance: This study is supported by MD Anderson Cancer Center Orlando (RB); Run Way to Hope and Hyundai Hope on Wheels Scholar grant awarded to DE). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 277. doi:1538-7445.AM2012-277

Abstract 3850: Production of recombinant lunasin peptides with enhanced anticancer activity using transient expression in tobacco

Abstract Epidemiological observations suggest a correlation between high levels of soybean product consumption and lowered incidence and mortality due to prostate, breast and colon cancer. Lunasin, a 43-44 amino acid peptide derived from soybean, has been implicated as a significant source of this anticancer activity. Lunasin contains nine consecutive aspartic acid residues at the C-terminus that bind to hypoacetlyated core histones, a minimal internal RGD cell adhesion motif and a helical region exhibiting structural homology to conserved sequences of chromatin binding proteins. Initial studies demonstrated that lunasin can prevent the transformation of mammalian cells by chemical carcinogens or viral oncogenes, and inhibits chemically-induced tumors in a mouse skin cancer model. These results suggest that lunasin may be useful as a cancer chemoprevention agent. More recent studies have demonstrated that lunasin can inhibit the proliferation of several established human cancer cell lines in vitro and in vivo, suggesting that lunasin may also be useful as a cancer therapeutic. Lunasin's anticancer effects are currently limited to specific cancer cell types and require 10-100 µM concentrations of peptide. We have now established a transient expression system based on the Tobacco Mosaic Virus vector, Geneware®, for large-scale production of modified forms of recombinant lunasin in tobacco. Our initial studies revealed that we could not detect any significant protein accumulation using constructs expressing lunasin peptides alone. We modified our strategy by expressing lunasin peptides as C-terminal fusions to green fluorescent protein (GFP) that includes a linker sequence containing a thrombin cleavage site. We have demonstrated that this system can produce GFP-lunasin at levels >100 mg/kg fresh weight tissue and demonstrated that the lunasin peptide can be recovered by proteolytic cleavage with thrombin. We have used this system to produce a modified form of lunasin that contains an N-terminal RGD domain sequence that preferentially binds to the αvβ6 integrin that is highly expressed in a number of epithelial-derived carcinomas. In vitro studies using cancer cell lines expressing the αvβ6 integrin demonstrate that the RGD-lunasin peptide is >10-fold more active in inhibiting cancer cell proliferation than the natural form of lunasin. Moreover, RGD-lunasin inhibits the proliferation of some cancer cell lines that are insensitive to 100 µM lunasin. These results demonstrate the feasibility of utilizing plant-based expression to produce more efficacious forms of lunasin that are targeted specifically to cancer cells and indicate that RGD lunasin may be useful as a cancer therapeutic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3850. doi:1538-7445.AM2012-3850

Abstract 3379: Adaptation of soft agar colony formation assays to identify agents which block proliferation of putative colon cancer stem cells

Abstract Cancer stem cells have been the focus of renewed interest in recent years. In theory these cells have the potential to limit the success of therapy, serving as therapy-resistant reservoirs of disease. In a previous study (Proc AACR 2011 #5202), each of seven human colon cancer cell lines was found to contain a subset of single cells, each of which possesses putative cancer stem cell properties: the capacity (a) to initiate and sustain highly prolific clonal growth in vitro as well as (b) to form tumor xenografts which are readily transplantable in vivo. Despite the extensive in vitro propagation of each human colon cancer cell line established from patient tumors many years ago, they have retained the functional capacity to initiate and sustain cancer cell growth from single cells both in vitro and in vivo. Because soft agar cultures require the clonal growth of single cells and permit the detection of stem-cell derived colonies on the basis of cumulative colony masses, we explored the utility of low cell inoculation density cultures and the stratification of colony data into multiple size categories as a means to identify agents which modulate cancer stem cell function. In the current study, HCT-116 and HT-29 parent cell lines were employed to evaluate the activity of selected differentiating agents, a stem-cell inducing agent, cytotoxic agents, and a battery of clinical and experimental therapeutic agents postulated to modulate cancer stem cell function in a three-step strategy: (1) testing each agent in 96-well plate colony formation assays; (2) a comparison of the indices of drug activity in colony formation assays with indices of drug activity measured in the NCI60 screen and identification of agents which are more active in blocking colony formation than inhibiting monolayer culture growth; (3) an evaluation of the highly active agents in a conventional 35-mm dish colony formation assay in order to quantify putative anti-cancer stem cell activity. Of 41 agents tested, 11 agents were observed to exhibit prominent effects upon colony formation at concentrations which were ½ - 1 log less than that required for the inhibition of growth in monolayer cultures. In contrast to cytotoxic agents which indiscriminately impact the growth of all colony categories, these 11 agents were differentially active against stem-cell derived colonies. Moreover, it is noteworthy that several clinical agents were highly active following long exposure to fractions of concentrations achievable in human patients. These results suggest that the soft agar colony formation assay of established human cancer cell lines in which cell inoculation and analysis parameters are optimized for the measurement of long-term growth in low cell inoculation density cultures provides a sensitive method for detecting agents which impact putative cancer stem cell function. (Supported in part by NCI Contract No. HHSN261200800001E) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3379. doi:1538-7445.AM2012-3379

Abstract 2067: Putative role of Nrf2 as a tumor suppressor through inhibition of angiogenesis

Abstract An increase in reactive oxygen species (ROS) is a common biochemical change in cancer cells. To investigate the mechanisms of ROS accumulation during the process of tumorigenesis we employed a well-characterized model of stepwise human mesenchymal stem cell (MSC) transformation. Transformed MSC (tMSC) became dependent upon oxidative stress, as treatment with antioxidants impaired their tumorigenic potential. The increased ROS in tMSC was a consequence of a striking global down-regulation of the cellular antioxidant machinery correlating with reduced levels of the transcription factor Nrf2, a key regulator of the cellular antioxidant response. Expression of constitutive active versions of oncogenic H-Ras, Raf and PI3K/AKT led to decreased levels of Nrf2 in non-transformed MSC. Down-regulation of Nrf2 plays a central role in the defective ROS-scavenging capacity and oncogenic potential of tMSC, since its over-expression induces antioxidants, reduces the levels of ROS and impairs in vivo tumor growth. Mechanistically we show that restoration of Nrf2 expression sensitizes transformed cells to apoptosis and reduces an angiogenic response. Elevated Nrf2, in turn, led to destabilization of HIF-1α and decreased expression of angiogenic factors such as adrenomedullin and VEGF during hypoxic conditions. Moreover, we found a significant reduction in both proliferation and sprouting of human umbilical vein endothelial cells after being cultured in hypoxic/conditioned media from tMSC over-expressing Nrf2 compared with control. Down-regulation of Nrf2 during human cell transformation is not restricted to MSC as Nrf2 expression was also down-regulated when human mammary epithelial cells (HMEC) were genetically modified and transformed with the same oncogenic elements used for MSC. We performed microarray comparison studies and observed that expression of Nrf2 was down-regulated in a panel of human tumors. Furthermore, expression of Nrf2 and its downstream gene NQO1 was enhanced in breast cancer cell lines such as MCF-7 and MDA-MB-231 when the cells were treated with U0126, an inhibitor of ERK1/2 pathway. These results suggest that down-regulation of Nrf2 expression through activation of Ras/Raf/ERK pathway is not unique to in vitro transformed MSC or HMEC, but also occurs in established human breast cancer cell lines. Overall, our results indicate that Nrf2 expression is reduced in certain human tumor cells by mechanisms that involve activation of Ras signaling, and reveal a new role for Nrf2 as a tumor suppressor through inhibition of angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2067. doi:1538-7445.AM2012-2067

Establishment and characterization of a novel chordoma cell line: CH22

Chordoma is a rare primary malignant bone tumor and there exist only a few established human chordoma cell lines. The scarcity of robust chordoma cell lines has limited the ability to study this tumor. In this report, we describe the establishment of a novel chordoma cell line and characterize its in vitro and in vivo behaviors. The tumor tissue was isolated from a patient with recurrent chordoma of the sacrum. After 6 months in culture, the chordoma cell line, referred here as CH22, was established. Microscopic analysis of two-dimensional culture confirmed that the CH22 cells exhibited a typical vacuolated cytoplasm similar to the well-established chordoma cell line U-CH1. Electron microscopy showed cohesive cells with numerous surface filopodia, pockets of glycogen and aggregates of intermediate tonofilaments in cytoplasm. Three-dimensional culture revealed that the CH22 cells could grow and form clusters by day 8. The MTT assays demonstrated that, compared with sensitive osteosarcoma cell lines, CH22 cells were relatively resistant to conventional chemotherapeutic drugs. Western blotting and immunofluorescence analysis confirmed that the CH22 cells expressed brachyury, vimentin, and cytokeratin. Finally, histological analysis of CH22 xenograft tumor tissues demonstrated the appearance of physaliphorous cells and positive staining of brachyury, cytokeratin, and S100. By CT and MRI, imaging xenografts showed the typical appearances seen in human chordomas. These findings suggest that the established novel human chordoma cell line CH22 and its tumorigenecity in SCID nude mice may serve as an important model for studying chordoma cell biology and the development of new therapeutic modalities.

Wnt/β-Catenin Signaling Regulates Yes-associated Protein (YAP) Gene Expression in Colorectal Carcinoma Cells

Mutations in the Wnt/β-catenin pathway occur in most colorectal cancers (CRCs), and these mutations lead to increased nuclear accumulation of the β-catenin transcriptional co-activator. In the nucleus, β-catenin associates with TCF/LEF sequence specific transcription factors to activate target gene expression. The Hippo pathway restricts cellular growth by preventing nuclear accumulation of the Yes-associated protein (YAP) transcriptional co-activator. YAP expression is elevated in CRCs suggesting that, like Wnt/β-catenin signaling, the Hippo pathway may contribute to colorectal carcinogenesis. Regulation of YAP at the post-translational level has been well studied but the transcription factors that control YAP gene expression are unknown. Here we demonstrate that β-catenin/TCF4 complexes bind a DNA enhancer element within the first intron of the YAP gene to drive YAP expression in CRC cells. As such, reducing β-catenin expression in CRC cells using shRNAs leads to decreased YAP mRNA and protein levels. YAP is abundantly expressed in the cytoplasm and nuclei of several established human colon cancer cell lines and this localization pattern is insensitive to plating density. Finally, we show that YAP expression is elevated in the majority of a panel of primary human colorectal tumors compared with its expression in uninvolved colonic mucosa, and that YAP and β-catenin localize to the nuclear compartment of tumor cells. Together, these results implicate YAP as an oncogene whose expression is driven by aberrant Wnt/β-catenin signaling in human CRC cells.

Paradoxical cross-talk between the Stat3 and MAPK pathways in CCL25-CCR9 mediated pancreatic cancer growth and proliferation.

231 Background: Chemokine receptors have been shown to regulate the progression of several malignancies. Signal transducer and activator of transcription 3 (Stat3) may contribute to the invasive phenotype of several human cancers, but its upstream signals have not been well characterized. Our objective was to investigate the relationship between the CCL25-CCR9 axis and Stat3 signaling in pancreatic cancer cells. Methods: We exposed two established human pancreatic cancer cell lines PANC-1 and MIAPaCa-2 to the cytokine CCL25 (800 ug/uL for 20 min) and measured the activation of Stat3 (phosho-Stat3) by Western blot assay. Stattic, a small molecule Stat3 inhibitor, was used (20uM) to antagonize Stat3 signaling. We also measured activation level of extracellular signal-regulated kinase (phospho-ERK) following CCL25 exposure and used UO126 (10uM), a small molecule MEK inhibitor, to antagonize the MAPK pathway. Changes in cell proliferation were measured by CellTiter Glo Fluorescence assay. Results: Constitutive phosphorylation of Stat3 was observed in both pancreatic cancer cell lines. Exposure of MIAPaCa-2 to CCL25 further increased phospho-Stat3 levels on Western blot assay. We also observed a concomitant increase in phospho-ERK levels with exposure to CCL25 in both cell lines. Exposure of pancreatic cancer cells to CCL25 significantly increased cell proliferation. To determine the mechanism of CCR9-mediated cell proliferation, we used stattic and UO126 to specifically inhibit Stat3 and MEK activation, respectively. Interestingly, pre-treatment with stattic prior to CCL25 exposure resulted in a paradoxical enhancement of phospho-ERK levels. Conversely, inhibition of the MAPK pathway with UO126 led to a paradoxical enhancement of phospho-Stat3 levels. Conclusions: Our results demonstrate that CCL25 activates Stat3 and MAPK pathways to contribute to pancreatic cancer proliferation. We also show potential cross-talk between Stat3 and MAPK pathways, wherein antagonism of one pathway resulted in paradoxical activation of the other pathway. Our findings suggest that therapeutic targeting of downstream pathways may require a multi-drug approach.

Permissiveness of human hepatoma cell lines for HCV infection

BackgroundAlthough primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread).ResultsWe found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection.ConclusionsWe conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection.

Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis

To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion, 8-hydroxyguanine (8OHG), in human cells. p.R154H, p.M255V, p.L360P, and p.P377L MUTYH variants, which were previously found in patients with colorectal polyposis and cancer, were selected for use in this study. Human H1299 cancer cell lines inducibly expressing wild-type (WT) MUTYH (type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system, enabling the genomic integration of the transposon sequence for MUTYH expression. MUTYH expression was examined after cumate induction using Western blotting analysis and immunofluorescence analysis. The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined. Next, the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay. The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate. All of the MUTYH variants and WT MUTYH were localized in the nucleus, and nuclear localization was also observed for FLAG-tagged MUTYH. The mutation frequency of supF was 2.2 × 10(-2) in the 8OHG-containing pMY189 plasmid and 2.5 × 10(-4) in WT pMY189 in empty vector cells, which was an 86-fold increase with the introduction of 8OHG. The mutation frequency (4.7 × 10(-3)) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells (P < 0.01). However, the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H, p.M255V, p.L360P, or p.P377L MUTYH variant were 1.84 × 10(-2), 1.55 × 10(-2), 1.91 × 10(-2), and 1.96 × 10(-2), respectively, meaning that no significant difference was observed in the mutation frequency between the empty vector cells and cells overexpressing MUTYH mutants. The suppressive activities of p.R154H, p.M255V, p.L360P, and p.P377L MUTYH variants against mutations caused by 8OHG are thought to be severely impaired in human cells.

Tolfenamic acid inhibits neuroblastoma cell proliferation and induces apoptosis: A novel therapeutic agent for neuroblastoma

Current therapeutic options for recurrent neuroblastoma have poor outcomes that warrant the development of novel therapeutic strategies. Specificity protein (Sp) transcription factors regulate several genes involved in cell proliferation, survival, and angiogenesis. Sp1 regulates genes believed to be important determinants of the biological behavior of neuroblastoma. Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to induce the degradation of Sp proteins and may serve as a novel anti-cancer agent. The objective of this investigation was to examine the anti-cancer activity of TA using established human neuroblastoma cell lines. We tested the anti-proliferative effect of TA using SH-SY5Y, CHLA90, LA1 55n, SHEP, Be2c, CMP 13Y, and SMS KCNR cell lines. Cells were treated with TA (0/25/50/100 µM) and cell viability was measured at 24, 48, and 72 h post-treatment. Selected neuroblastoma cell lines were treated with 50 µM TA for 24 and 48 h and tested for cell apoptosis using Annexin-V staining. Caspase activity was measured with caspase 3/7 Glo kit. Cell lysates were prepared and the expression of Sp1, survivin, and c-PARP were evaluated through Western blot analysis. TA significantly inhibited the growth of neuroblastoma cells in a dose/time-dependent manner and significantly decreased Sp1 and survivin expression. Apart from cell cycle (G0/G1) arrest, TA caused significant increase in the apoptotic cell population, caspase 3/7 activity, and c-PARP expression. These results show that TA effectively inhibits neuroblastoma cell growth potentially through suppressing mitosis, Sp1, and survivin expression, and inducing apoptosis. These results show TA as a novel therapeutic agent for neuroblastoma.

Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma

Despite radical surgery and multi-agent chemotherapy, less than one third of patients with recurrent or metastatic osteosarcoma (OS) survive. The limited efficacy of current therapeutic approaches to target tumor-initiating cells (TICs) may explain this dismal outcome. The purpose of this study was to assess the impact of modified T cells expressing a human epidermal growth factor receptor (HER2)-specific chimeric antigen receptor in the OS TIC compartment of human established cell lines. Using the sarcosphere formation assay, we found that OS TICs were resistant to increasing methotrexate concentrations. In contrast, HER2-specific T cells decreased markedly sarcosphere formation capacity and the ability to generate bone tumors in immunodeficient mice after orthotopic transplantation. In vivo, administration of HER2-specific T cells significantly reduced TICs in bulky tumors as judged by decreased sarcosphere forming efficiency in OS cells isolated from explanted tumors. We demonstrate that HER2-specific T cells target drug resistant TICs in established OS cell lines, suggesting that incorporating immunotherapy into current treatment strategies for OS has the potential to improve outcomes.

Boswellia sacra essential oil induces tumor cell-specific apoptosis and suppresses tumor aggressiveness in cultured human breast cancer cells

BackgroundGum resins obtained from trees of the Burseraceae family (Boswellia sp.) are important ingredients in incense and perfumes. Extracts prepared from Boswellia sp. gum resins have been shown to possess anti-inflammatory and anti-neoplastic effects. Essential oil prepared by distillation of the gum resin traditionally used for aromatic therapy has also been shown to have tumor cell-specific anti-proliferative and pro-apoptotic activities. The objective of this study was to optimize conditions for preparing Boswellea sacra essential oil with the highest biological activity in inducing tumor cell-specific cytotoxicity and suppressing aggressive tumor phenotypes in human breast cancer cells.MethodsBoswellia sacra essential oil was prepared from Omani Hougari grade resins through hydrodistillation at 78 or 100 oC for 12 hours. Chemical compositions were identified by gas chromatography-mass spectrometry; and total boswellic acids contents were quantified by high-performance liquid chromatography. Boswellia sacra essential oil-mediated cell viability and death were studied in established human breast cancer cell lines (T47D, MCF7, MDA-MB-231) and an immortalized normal human breast cell line (MCF10-2A). Apoptosis was assayed by genomic DNA fragmentation. Anti-invasive and anti-multicellular tumor properties were evaluated by cellular network and spheroid formation models, respectively. Western blot analysis was performed to study Boswellia sacra essential oil-regulated proteins involved in apoptosis, signaling pathways, and cell cycle regulation.ResultsMore abundant high molecular weight compounds, including boswellic acids, were present in Boswellia sacra essential oil prepared at 100 oC hydrodistillation. All three human breast cancer cell lines were sensitive to essential oil treatment with reduced cell viability and elevated cell death, whereas the immortalized normal human breast cell line was more resistant to essential oil treatment. Boswellia sacra essential oil hydrodistilled at 100 oC was more potent than the essential oil prepared at 78 oC in inducing cancer cell death, preventing the cellular network formation (MDA-MB-231) cells on Matrigel, causing the breakdown of multicellular tumor spheroids (T47D cells), and regulating molecules involved in apoptosis, signal transduction, and cell cycle progression.ConclusionsSimilar to our previous observations in human bladder cancer cells, Boswellia sacra essential oil induces breast cancer cell-specific cytotoxicity. Suppression of cellular network formation and disruption of spheroid development of breast cancer cells by Boswellia sacra essential oil suggest that the essential oil may be effective for advanced breast cancer. Consistently, the essential oil represses signaling pathways and cell cycle regulators that have been proposed as therapeutic targets for breast cancer. Future pre-clinical and clinical studies are urgently needed to evaluate the safety and efficacy of Boswellia sacra essential oil as a therapeutic agent for treating breast cancer.