Modified Papanicolaou staining for oral swab samples stored long term.

Pathologists collect swab samples for Papanicolaou (Pap) staining to diagnose various diseases including cancer and HIV. Time constraints and limited resources, may preclude staining a sample immediately. To re-confirm results, samples must be frozen for later analysis. We present a method for Pap staining cells that have been stored long term. An effective method for Pap staining of frozen cells should enable flexibility for processing samples.

Claudin proteins, outside-in signaling, and carcinogenesis.

Environment affects an individual's development and disease risk which then suggest that the environmental cues must have ways of reaching to the cellular nuclei to orchestrate desired genetic changes. Polarized and differentiated epithelial cells join together by cell-cell adhesions to create a protective sheet which separates body's internal milieu from its environment, albeit in highly regulated manner. Among these cell-cell adhesions, a key role of tight junction, the apical cell-cell adhesion, in maintaining epithelial cell polarity and differentiation is well recognized. Moreover, significant changes in expression and cellular distribution of claudin proteins, integral component of the tight junction, characterize pathophysiological changes including neoplastic growth and progression. Studies have further confirmed existence of complex claudin-based interactomes and demonstrated that changes in such protein partnering can influence barrier integrity and communication between a cell and its environment to produce undesired outcome. Cell signaling is the process by which cells respond to their environment to make dynamic decisions to live, grow and proliferate, or die. Thus, pivotal role of the deregulated tight junction structure/function in influencing cellular signaling cascades to alter cellular phenotype can be envisaged, however, is not well understood. Needless to mention that advanced knowledge in this area can help improve therapeutic considerations and preventive measures. Here, we discuss potential role of the tight junction in the regulation of "outside-in" signaling to regulate cancer growth, with specific focus upon the claudin family of proteins.

Claudin-1 promotes TNF-α-induced epithelial-mesenchymal transition and migration in colorectal adenocarcinoma cells.

Epithelial-mesenchymal transition (EMT) is an important mechanism in cancer progression and malignancy including colorectal cancer (CRC). Importantly, inflammatory mediators are critical constituents of the local tumor environment and an intimate link between CRC progression and inflammation is now validated. We and others have reported key role of the deregulated claudin-1 expression in colon carcinogenesis including colitis-associated colon cancer (CAC). However, the causal association between claudin-1 expression and inflammation-induced colon cancer progression remains unclear. Here we demonstrate, TNF-α, a pro-inflammatory cytokine, regulates claudin-1 to modulate epithelial to mesenchymal transition (EMT) and migration in colon adenocarcinoma cells. Importantly, colon cancer cells cultured in the presence of TNF-α (10ng/ml), demonstrated a sharp decrease in E-cadherin expression and an increase in vimentin expression (versus control cells). Interestingly, TNF-α treatment also upregulated (and delocalized) claudin-1 expression in a time-dependent manner accompanied by increase in proliferation and wound healing. Furthermore, similar to our previous observation that claudin-1 overexpression in CRC cells induces ERK1/2 and Src- activation, signaling associated with colon cancer cell survival and transformation, TNF-α-treatment induced upregulation of phospho-ERK1/2 and -Src expression. The shRNA-mediated inhibition of claudin-1 expression largely abrogated the TNF-α-induced changes in EMT, proliferation, migration, p-Erk and p-Src expression. Taken together, our data demonstrate TNF-α mediated regulation of claudin-1 and tumorigenic abilities of colon cancer cells and highlights a key role of deregulated claudin-1 expression in inflammation-induced colorectal cancer growth and progression, through the regulation of the ERK and Src-signaling.

Oligonucleotides and G-quadruplex stabilizers: targeting telomeres and telomerase in cancer therapy.

Cancer is a leading cause of death worldwide and an estimated 1 in 4 deaths in the United States is due to cancer. Despite recent advances in cancer treatment, adverse effects related to cancer therapy remain a limiting factor for many patients. The ideal cancer treatment would selectively target cancerous cells while sparing normal, healthy cells to offer maximal therapeutic benefit while minimizing toxicity. Telomeres are structurally unique DNA sequences at the end of human chromosomes, which play an integral role in the cellular mortality of normal cells. As telomeres shorten with successive cellular divisions, cells develop chromosomal instability and undergo either apoptosis or senescence. In many cancers, this apoptosis or senescence is avoided as normal telomere length is maintained by a ribonucleoprotein reverse transcriptase called telomerase. Telomerase is expressed in more than 85% of all cancers and confers cancerous cells with a replicative immortality, which is a hallmark of malignant tumors. In contrast, telomerase activity is not detectable in the majority of normal somatic cell populations. Therefore, the targeting of telomerase and telomere maintenance mechanisms represent a potentially promising therapeutic approach for various types of cancer. This review evaluates the roles of GRN163L, T-oligo and small molecule G-quadruplex stabilizers as potential anticancer therapies by targeting telomerase and other telomere maintenance mechanisms.

Targeting c-Met in melanoma: mechanism of resistance and efficacy of novel combinatorial inhibitor therapy.

Numerous tyrosine kinase inhibitors (TKIs) targeting c-Met are currently in clinical trials for several cancers. Their efficacy is limited due to the development of resistance. The present study aims to elucidate this mechanism of c-Met TKI resistance by investigating key mTOR and Wnt signaling proteins in melanoma cell lines resistant to SU11274, a c-Met TKI. Xenografts from RU melanoma cells treated with c-Met TKIs SU11274 and JNJ38877605 showed a 7- and 6-fold reduction in tumor size, respectively. Resistant cells displayed upregulation of phosphorylated c-Met, mTOR, p70S6Kinase, 4E-BP1, ERK, LRP6, and active ß-catenin. In addition, GATA-6, a Wnt signaling regulator, was upregulated, and Axin, a negative regulator of the Wnt pathway, was downregulated in resistant cells. Modulation of these mTOR and Wnt pathway proteins was also prevented by combination treatment with SU11274, everolimus, an mTOR inhibitor, and XAV939, a Wnt inhibitor. Treatment with everolimus, resulted in 56% growth inhibition, and a triple combination of SU11274, everolimus and XAV939, resulted in 95% growth inhibition in RU cells. The V600E BRAF mutation was found to be positive only in MU cells. Combination treatment with a c-Met TKI and a BRAF inhibitor displayed a synergistic effect in reducing MU cell viability. These studies indicate activation of mTOR and Wnt signaling pathways in c-Met TKI resistant melanoma cells and suggest that concurrent targeting of c-Met, mTOR, and Wnt pathways and BRAF may improve efficacy over traditional TKI monotherapy in melanoma patients.

T-oligo as an anticancer agent in colorectal cancer.

In the United States, there will be an estimated 96,830 new cases of colorectal cancer (CRC) and 50,310 deaths in 2014. CRC is often detected at late stages of the disease, at which point there is no effective chemotherapy. Thus, there is an urgent need for effective novel therapies that have minimal effects on normal cells. T-oligo, an oligonucleotide homologous to the 3'-telomere overhang, induces potent DNA damage responses in multiple malignant cell types, however, its efficacy in CRC has not been studied. This is the first investigation demonstrating T-oligo-induced anticancer effects in two CRC cell lines, HT-29 and LoVo, which are highly resistant to conventional chemotherapies. In this investigation, we show that T-oligo may mediate its DNA damage responses through the p53/p73 pathway, thereby inhibiting cellular proliferation and inducing apoptosis or senescence. Additionally, upregulation of downstream DNA damage response proteins, including E2F1, p53 or p73, was observed. In LoVo cells, T-oligo induced senescence, decreased clonogenicity, and increased expression of senescence associated proteins p21, p27, and p53. In addition, downregulation of POT1 and TRF2, two components of the shelterin protein complex which protects telomeric ends, was observed. Moreover, we studied the antiproliferative effects of T-oligo in combination with an EGFR tyrosine kinase inhibitor, Gefitinib, which resulted in an additive inhibitory effect on cellular proliferation. Collectively, these data provide evidence that T-oligo alone, or in combination with other molecularly targeted therapies, has potential as an anti-cancer agent in CRC.

Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy.

Oligonucleotides homologous to 3'-telomere overhang (T-oligos) trigger inherent telomere-based DNA damage responses mediated by p53 and/or ATM and induce senescence or apoptosis in various cancerous cells. However, T-oligo has limited stability in vivo due to serum and intracellular nucleases. To develop T-oligo as an innovative, effective therapeutic drug and to understand its mechanism of action, we investigated the antitumor effects of T-oligo or T-oligo complexed with a novel cationic alpha helical peptide, PVBLG-8 (PVBLG), in a p53 null melanoma cell line both in vitro and in vivo. The uptake of T-oligo by MM-AN cells was confirmed by immunofluorescence, and fluorescence-activated cell sorting analysis indicated that the T-oligo-PVBLG nanocomplex increased uptake by 15-fold. In vitro results showed a 3-fold increase in MM-AN cell growth inhibition by the T-oligo-PVBLG nanocomplex compared with T-oligo alone. Treatment of preformed tumors in immunodeficient mice with the T-oligo-PVBLG nanocomplex resulted in a 3-fold reduction in tumor volume compared with T-oligo alone. This reduction in tumor volume was associated with decreased vascular endothelial growth factor expression and induction of thrombospondin-1 expression and apoptosis. Moreover, T-oligo treatment downregulated procaspase-3 and procaspase-7 and increased catalytic activity of caspase-3 by 4-fold in MM-AN cells. Furthermore, T-oligo induced a 10-fold increase of senescence and upregulated the melanoma tumor-associated antigens MART-1, tyrosinase, and thrombospondin-1 in MM-AN cells, which are currently being targeted for melanoma immunotherapy. Interestingly, siRNA-mediated knockdown of p73 (4-10-fold) abolished this upregulation of tumor-associated antigens. In summary, we suggest a key role of p73 in mediating the anticancer effects of T-oligo and introduce a novel nanoparticle, the T-oligo-PVBLG nanocomplex, as an effective anticancer therapeutic.

Alternative signaling pathways as potential therapeutic targets for overcoming EGFR and c-Met inhibitor resistance in non-small cell lung cancer.

The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC50 concentrations of resistant lines exhibited a 4-5 and 11-22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2-4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.

hnRNP C1/C2 and Pur-beta proteins mediate induction of senescence by oligonucleotides homologous to the telomere overhang.

BACKGROUND: Experimental disruption of the telomere overhang induces a potent DNA damage response and is the target of newly emerging cancer therapeutics. Introduction of T-oligo, an eleven-base oligonucleotide homologous to the 3'-telomeric overhang, mimics telomere disruption and induces DNA damage responses through activation of p53, p73, p95/Nbs1, E2F1, pRb, and other DNA damage response proteins. ATM (ataxia telangiectasia mutated) was once thought to be the primary driver of T-oligo-induced DNA damage responses; however, recent experiments have highlighted other key proteins that may also play a significant role. METHODS: To identify proteins associated with T-oligo, MM-AN cells were treated with biotinylated T-oligo or complementary oligonucleotide, cell lysates were run on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and the protein bands observed after treatment of cells with T-oligo or complementary oligonucleotide were analyzed using mass spectrometry. To study the effect of T-oligo on expression of hnRNP C1/C2 (heterogeneous nuclear ribonucleoprotein C1 and C2) and purine-rich element binding proteins (Pur proteins), cells were treated with T-oligo, and immunoblotting experiments were performed. To determine their role in senescence, cells were treated with shRNA (short hairpin ribonucleic acid) against these proteins, and senescence was studied using the senescence associated beta-galactosidase assay. RESULTS: Using mass spectrometry, RNA-binding hnRNP C1/C2 and DNA-binding Pur proteins were found to associate with T-oligo. hnRNP C1/C2 exhibited increased expression (3.6-12.0-fold) in non-small-cell lung cancer (NSCLC) and in melanoma cells (4.5-5.2-fold), and Pur proteins exhibited increased expression of 2.2-fold in NSCLC and 2.0-fold in melanoma cells after T-oligo treatment. Experimental knockdown of hnRNP C1/C2 and Pur-beta completely abrogated T-oligo induced senescence in both MU melanoma and H358 NSCLC cells. Additionally, knockdown of Pur-beta prevented T-oligo-induced phosphorylation of p53, hypophosphorylation of pRb, and upregulation of E2F1, p21, and p53. CONCLUSION: These novel findings highlight proteins essential to T-oligo's anticancer effects that may be of interest in telomere biology and cancer therapeutics.