RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin

Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 mu M, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 mu M. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

A side order of stem cells: The SP phenotype

A defining property of murine hematopoietic stein cells (HSCs) is low fluorescence after staining with Hoechst 33342 and Rhodamine 123. These dyes have proven to be remarkably powerful tools in the purification and characterization of HSCs when used alone or in combination with antibodies directed against stem cell epitopes. Hoechst low cells are described as side population (SP) cells by virtue of their typical profiles in Hoechst red versus Hoechst blue bivariate fluorescent-activated cell sorting dot plots. Recently, excitement has been generated by the findings that putative stem cells from solid tissues may also possess this SP phenotype. SP cells have now been isolated from a wide variety of mammalian tissues based on this same dye efflux phenomenon, and in many cases this cell population has been shown to contain apparently multipotent stem cells. What is yet to be clearly addressed is whether cell fusion accounts for this perceived SP multipotency. Indeed, if low fluorescence after Hoechst staining is a phenotype shared by hematopoietic and organ-specific stem cells, do all resident tissue SP cells have bone marrow origins or might the SP phenotype be a property common to all stem cells? Subject to further analysis, the SP phenotype may prove invaluable for the initial isolation of resident tissue stem cells in the absence of definitive cell-surface markers and may have broad-ranging applications in stem cell biology, from the purification of novel stem cell populations to the development of autologous stem cell therapies.

Cell surface expression of urokinase receptor in normal mammary epithelial cells and breast cancer cell lines

Background: The urokinase receptor (uPAR) is important in the process of extracellular matrix degradation occurring during cancer cell invasion and metastasis. We wished to quantify uPAR on the surfaces of normal mammary epithelial cells (HMEC) and 6 well-known breast cancer cell lines using flow cytometry. Materials and Methods: Cell surface uPAR was labelled with a monoclonal antibody, and this was detected with a florescent-labelled second antibody and accurately measured using flow cytometry. The measured fluorescent signals of the stained cells were interpolated with those of Quantum Simply Cellular bead standards to determine the number of uPAR sites per cell. Results: The breast cancer cell lines ranged from 13,700 to 50,800 uPAR sites per cell, whilst HMEC cells had only 2,500 sites. Conclusions: This simple and reliable method showed that the expression of cell surface uPAR is higher in the breast cancer cell lines than in the normal mammary cells.

Fas-FasL expression and interactions in mouse tumor cell lines: Implications for tumor immune escape

The Fas system, comprising the Fas receptor (Fas/Apo-1/CD95) and its ligand, Fas ligand (FasL), is a central mediator of programmed cell death in various physiological and pathological processes. FasL exists as transmembrane and soluble forms and induces apoptosis on crosslinking with Fas receptor. Recent evidence indicated that tumor cells exploit this system for their immunologic escape that includes the loss of Fas and the gain of FasL expression. In the present study, nine mouse tumor cell lines of diverse origin were examined immunocytochemically for the expression of Fas and FasL. Nine of nine cell lines expressed FasL, and five of nine cell lines expressed Fas. FasL expression in these tumor cell lines was demonstrated to be functional by its induction of apoptosis in Fas-sensitive target cells in coculture experiments. These results suggest that FasL may be a prevalent mediator of immune privilege in mouse malignancies, and support the recently proposed "counterattack model" for local elimination of tumor-reactive immune cells by tumor cell-derived FasL.^ Culture supernatant of four cell lines expressing FasL showed cytotoxic effect on Fas-sensitive target cells, indicating the possibility of secreted FasL in the medium. The Fas-expressing cell lines were sensitized to anti-Fas antibody cytotoxicity following treatment with IL-2 and IFN-$\gamma$, suggesting cytokine stimulation as an effective target for future immunotherapeutic strategies. ^

Structural and Functional Analysis of the Caspase –dependent and –independent Domains of the X-linked Inhibitor of Apoptosis Protein in Inflammatory Breast Cancer Tumor Biology

Inflammatory breast cancer (IBC) is an extremely rare but highly aggressive form of breast cancer characterized by the rapid development of therapeutic resistance leading to particularly poor survival. Our previous work focused on the elucidation of factors that mediate therapeutic resistance in IBC and identified increased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), to correlate with the development of resistance to chemotherapeutics. Although XIAP is classically thought of as an inhibitor of caspase activation, multiple studies have revealed that XIAP can also function as a signaling intermediate in numerous pathways. Based on preliminary evidence revealing high expression of XIAP in pre-treatment IBC cells rather than only subsequent to the development of resistance, we hypothesized that XIAP could play an important signaling role in IBC pathobiology outside of its heavily published apoptotic inhibition function. Further, based on our discovery of inhibition of chemotherapeutic efficacy, we postulated that XIAP overexpression might also play a role in resistance to other forms of therapy, such as immunotherapy. Finally, we posited that targeting of specific redox adaptive mechanisms, which are observed to be a significant barrier to successful treatment of IBC, could overcome therapeutic resistance and enhance the efficacy of chemo-, radio-, and immuno- therapies. To address these hypotheses our objectives were: 1. to determine a role for XIAP in IBC pathobiology and to elucidate the upstream regulators and downstream effectors of XIAP; 2. to evaluate and describe a role for XIAP in the inhibition of immunotherapy; and 3. to develop and characterize novel redox modulatory strategies that target identified mechanisms to prevent or reverse therapeutic resistance.

Using various genomic and proteomic approaches, combined with analysis of cellular viability, proliferation, and growth parameters both in vitro and in vivo, we demonstrate that XIAP plays a central role in both IBC pathobiology in a manner mostly independent of its role as a caspase-binding protein. Modulation of XIAP expression in cells derived from patients prior to any therapeutic intervention significantly altered key aspects IBC biology including, but not limited to: IBC-specific gene signatures; the tumorigenic capacity of tumor cells; and the metastatic phenotype of IBC, all of which are revealed to functionally hinge on XIAP-mediated NFκB activation, a robust molecular determinant of IBC. Identification of the mechanism of XIAP-mediated NFκB activation led to the characterization of novel peptide-based antagonist which was further used to identify that increased NFκB activation was responsible for redox adaptation previously observed in therapy-resistant IBC cells. Lastly, we describe the targeting of this XIAP-NFκB-ROS axis using a novel redox modulatory strategy both in vitro and in vivo. Together, the data presented here characterize a novel and crucial role for XIAP both in therapeutic resistance and the pathobiology of IBC; these results confirm our previous work in acquired therapeutic resistance and establish the feasibility of targeting XIAP-NFκB and the redox adaptive phenotype of IBC as a means to enhance survival of patients.

Tyrosine kinase inhibitor insensitivity of non-cycling CD34+ human acute myeloid leukaemia cells with FMS-like tyrosine kinase 3 mutations.

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

Cationic Bovine Serum Albumin (CBA) conjugated Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles for extended delivery of methotrexate into brain tumor

Current treatment strategies for the treatment of brain tumor have been hindered primarily by the presence of highly lipophilic insurmountable blood-brain barrier (BBB). The purpose of current research was to investigate the efficiency of engineered biocompatible polymeric nanoparticles (NPs) as drug delivery vehicle to bypass the BBB and enhance biopharmaceutical attributes of anti-metabolite methotrexate (MTX) encapsulated NPs. The NPs were prepared by solvent diffusion method using cationic bovine serum albumin (CBA), and characterized for physicochemical parameters such as particle size, polydispersity index, and zeta-potential; while the surface modification was confirmed by FTIR, and NMR spectroscopy. Developed NPs exhibited zestful relocation of FITC tagged NPs across BBB in albino rats. Further, hemolytic studies confirmed them to be non-toxic and biocompatible as compared to free MTX. In vitro cytotoxicity assay of our engineered NPs on HNGC1 tumor cells proved superior uptake in tumor cells; and elicited potent cytotoxic effect as compared to plain NPs and free MTX solution. The outcomes of the study evidently indicate the prospective of CBA conjugated poly (D,L-lactide-co-glycolide) (PLGA) NPs loaded with MTX in brain cancer bomber with amplified capability to circumvent BBB.

Ibuprofen Inhibits Overexpression of Tumor-Related Rac1b through SRSF1

Abstract: The serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression. A previous analysis of colorectal tumors revealed that overexpression of splice variant Rac1b occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival. Patients with inflamed human colonic mucosa also have increased expression of Rac1b as well as mice with experimentally induced colitis. The increase of Rac1b in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen. Purpose: The objective of our study is to understand the molecular regulation of Rac1b alternative splicing event and how it contributes to tumorigenesis. Experimental description: HT29 colorectal cell line was used as model to test several signaling pathways after 48h of treatment with ibuprofen. For this we analyzed the proteins of interest by Western Blot and the transcript levels by RT-PCR. Results: Mechanistic studies in cultured HT29 colorectal tumor cells revealed that ibuprofen inhibited Rac1b expression in a cyclooxygenase inhibition–independent manner and targets directly the alternative splicing event. Here, we provide evidence that ibuprofen leads to a decrease in expression of SRSF1, a splicing factor that we previously identified to promote Rac1b alternative splicing. Together, our results suggest that stromal cues, namely, inflammation, can trigger changes in Rac1b expression in the colon and identify ibuprofen as a highly specific and efficient inhibitor of Rac1b overexpression in colorectal tumors. Conclusions: Our data identify an additional cyclooxygenase–independent action of ibuprofen and suggest it may be beneficial in the treatment of patients with the subtype of BRAF-mutated serrated colorectal tumors.

Septic Shock Sera Containing Circulating Histones Induce Dendritic Cell–Regulated Necrosis in Fatal Septic Shock Patients

International audience

The role of CD8+CCR4+ T-cells in axial spondyloarthritis

Axial spondyloarthritis (AxSpA) is an inflammatory disease affecting the axial skeleton. The infiltrate of T-cells in the structural lesions has been found to contribute to bone remodeling, but consensus relating the functional contribution of different T-cell subsets to pathogenesis has not been reached yet. Aim of the project was to characterize circulating T-cells and their homing markers from axSpA patients in order to identify cellular populations that could migrate to inflamed tissues and be implicated in axSpA. We found an altered proportion of circulating naïve and memory T-cells in axSpA patients, and a skew in favor of CD8+ T-cells expressing the chemokine receptor CCR4. Since CCL17 and CCL22, the two ligands for CCR4, are found to be elevated in the sera of axSpA patients, we investigated in details the role of CD8+CCR4+ T cells in axSpA. Our data showed that circulating CD8+CCR4+ T-cells display an effector memory phenotype and express homing markers for tissues that are target of the disease. Noteworthy, CD8+CCR4+ T cells from axSpA patients were activated, expressed markers of proliferation and acquired a cytotoxic phenotype, as demonstrated by the increased production of granzyme and perforin. CD8+CCR4+ T cells from axSpA patients upregulate the transcription of genes involved in bone mineralization and downregulate genes involved in osteoclast differentiation, indicating their possible involvement in bone remodeling. Furthermore, CD8+CCR4+ T cells stimulated with PMA and ionomycin were able to produce and release TNF and IL-8, two cytokines involved in osteoclastogenesis, indicating that CD8+CCR4+ T-cells after stimulation would be able to promote osteoclasts differentiation and neutrophils recruitment. Taken together our data suggest that CD8+CCR4+ T cells might exert a pathogenic role in axSpA, by releasing mediators of tissue damage, bone remodeling and recruitment of other pro inflammatory cells.

Antioxidant And Antiproliferative Activities Of Methanolic Extract From A Neglected Agricultural Product: Corn Cobs.

Neglected agricultural products (NAPs) are defined as discarded material in agricultural production. Corn cobs are a major waste of agriculture maize. Here, a methanolic extract from corn cobs (MEC) was obtained. MEC contains phenolic compounds, protein, carbohydrates (1.4:0.001:0.001). We evaluated the in vitro and in vivo antioxidant potential of MEC. Furthermore, its antiproliferative property against tumor cells was assessed through MTT assays and proteins related to apoptosis in tumor cells were examined by western blot. MEC showed no hydroxyl radical scavenger capacity, but it showed antioxidant activity in Total Antioxidant Capacity and DPPH scavenger ability assays. MEC showed higher Reducing Power than ascorbic acid and exhibited high Superoxide Scavenging activity. In tumor cell culture, MEC increased catalase, metallothionein and superoxide dismutase expression in accordance with the antioxidant tests. In vivo antioxidant test, MEC restored SOD and CAT, decreased malondialdehyde activities and showed high Trolox Equivalent Antioxidant Capacity in animals treated with CCl4. Furthermore, MEC decreased HeLa cells viability by apoptosis due an increase of Bax/Bcl-2 ratio, caspase 3 active. Protein kinase C expression increased was also detected in treated tumor cells. Thus, our findings pointed out the biotechnological potential of corn cobs as a source of molecules with pharmacological activity.

Antimicrobial And Antiproliferative Potential Of Anadenanthera Colubrina (vell.) Brenan.

The aim of the present study was to perform an in vitro analysis of the antimicrobial and antiproliferative potential of an extract from Anadenanthera colubrina (Vell.) Brenan (angico) and chemically characterize the crude extract. Antimicrobial action was evaluated based on the minimum inhibitory concentration (MIC), minimum bactericidal/fungicidal concentration, and the inhibition of formation to oral biofilm. Cell morphology was determined through scanning electron microscopy (SEM). Six strains of tumor cells were used for the determination of antiproliferative potential. The extract demonstrated strong antifungal activity against Candida albicans ATCC 18804 (MIC = 0.031 mg/mL), with similar activity found regarding the ethyl acetate fraction. The extract and active fraction also demonstrated the capacity to inhibit the formation of Candida albicans to oral biofilm after 48 hours, with median values equal to or greater than the control group, but the difference did not achieve statistical significance (P > 0.05). SEM revealed alterations in the cell morphology of the yeast. Regarding antiproliferative activity, the extract demonstrated cytostatic potential in all strains tested. The present findings suggest strong antifungal potential for Anadenanthera colubrina (Vell.) Brenan as well as a tendency toward diminishing the growth of human tumor cells.

Ki-1/57 And Cgi-55 Ectopic Expression Impact Cellular Pathways Involved In Proliferation And Stress Response Regulation.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

Galectin-3 Up-regulation In Hypoxic And Nutrient Deprived Microenvironments Promotes Cell Survival.

Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.