Response Of Carcinoma Cells Research Articles

Antitumor activity and transcriptome sequencing (RNA-seq) analyses of hepatocellular carcinoma cells in response to exposure triterpene-nucleoside conjugates

Fifteen betulonic/betulinic acid conjugated with nucleoside derivatives were synthesized to enhance antitumor potency and water solubility. Among these, the methylated betulonic acid-azidothymidine compound (8c) exhibited a broad-spectrum of antitumor activity against three tested tumor cell lines, including SMMC-7721 (IC50 = 5.02 μM), KYSE-150 (IC50 = 5.68 μM), and SW620 (IC50 = 4.61 μM) and along with lower toxicity (TC50 > 100 μM) estimated by zebrafish embryos assay. Compared to betulinic acid (<0.05 μg/mL), compound 8c showed approximately 40-fold higher water solubility (1.98 μg/mL). In SMMC-7721 cells, compound 8c induced autophagy and apoptosis as its concentration increased. Transcriptomic sequencing analysis was used to understand the potential impacts of the underlying mechanism of 8c on SMMC-7721 cells. Transcriptomic studies indicated that compound 8c could activate autophagy by inhibiting the PI3K/AKT pathway in SMMC-7721 cells. Furthermore, in the xenograft mice study, compound 8c significantly slowed down the tumor growth, as potent as paclitaxel treated group. In conclusion, methylated betulonic acid-azidothymidine compound (8c) not only increases water solubility, but also enhances the potency against hepatocellular carcinoma cells by inducing autophagy and apoptosis, and suppressing the PI3K/Akt/mTOR signaling pathway.

Silymarin Mediates Apoptosis Through Activation of JNK/ERK Signaling Pathway in Human Colon Carcinoma Cells in Response to ( 60 Co) Gamma Radiation

Silymarin Mediates Apoptosis Through Activation of JNK/ERK Signaling Pathway in Human Colon Carcinoma Cells in Response to ( 60 Co) Gamma Radiation

RNA Sequencing Demonstrates That Circular RNA Regulates Avian Influenza Virus Replication in Human Cells.

Circular RNAs (circRNAs) are involved in diverse biological processes. Avian influenza virus (AIV) can cross the species barrier to infect humans. Here, we employed RNA sequencing technology to profile circRNA, microRNA, and mRNA expression in human lung carcinoma cells in response to AIV or human influenza A virus (IAV) infection at viral replication. The analysis revealed that the expression of 475 common circRNAs were significantly regulated. The 381 and 1163 up-regulated circRNAs were induced by AIV at 8 and 16 h, respectively. Subsequently, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses were also conducted for the AIV-specific up-regulated circRNAs. Moreover, the circRNAs were characterized, of which six were verified by quantitative real-time PCR. We further confirmed that expression of the selected circRNAs only increased following AIV infection. Knocking down the selected circRNAs promoted AIV proliferation, and overexpression of three of the candidate circRNAs restricted AIV replication and proliferation. We further analyzed that AIV-specific up-regulated circRNA mechanisms might function through the ceRNA network to affect the “Endocytosis” pathway and the “Cell cycle process”. These data provide the first expression profile of AIV-specific up-regulated circRNAs and shed new light on the pathogenesis of AIV infection. Our findings also suggest that these circRNAs serve an important role in AIV infection.

ERRFI1 induces apoptosis of hepatocellular carcinoma cells in response to tryptophan deficiency

Tryptophan metabolism is an essential regulator of tumor immune evasion. However, the effect of tryptophan metabolism on cancer cells remains largely unknown. Here, we find that tumor cells have distinct responses to tryptophan deficiency in terms of cell growth, no matter hepatocellular carcinoma (HCC) cells, lung cancer cells, or breast cancer cells. Further study shows that ERRFI1 is upregulated in sensitive HCC cells, but not in resistant HCC cells, in response to tryptophan deficiency, and ERRFI1 expression level positively correlates with HCC patient overall survival. ERRFI1 knockdown recovers tryptophan deficiency-suppressed cell growth of sensitive HCC cells. In contrast, ERRFI1 overexpression sensitizes resistant HCC cells to tryptophan deficiency. Moreover, ERRFI1 induces apoptosis by binding PDCD2 in HCC cells, PDCD2 knockdown decreases the ERRFI1-induced apoptosis in HCC cells. Thus, we conclude that ERRFI1-induced apoptosis increases the sensitivity of HCC cells to tryptophan deficiency and ERRFI1 interacts with PDCD2 to induce apoptosis in HCC cells.

Recombinant BCG overexpressing STING agonist elicits trained immunity and improved antitumor efficacy in non-muscle invasive bladder cancer

BCG remains first-line therapy for non-muscle invasive bladder cancer (NIMBC) but its mechanism of action is not fully understood nor is it completely efficacious. We engineered a recombinant BCG (rBCG) that releases increased levels of STING agonist, c-di-AMP and compared effects of rBCG to wild type BCG (WT-BCG) by assessing antitumor efficacy and its ability to elicit trained immunity in NMIBC models. Rat MNU-carcinogen model of NMIBC was used to compare antitumor efficacy of 6 weekly intravesical installations of WT-BCG versus rBCG by pathologic tumor grading of bladder tissue and qRT-PCR for Th1 cytokines. Primary human and murine macrophage and urothelial carcinoma cell lines were treated with WT-BCG and rBCG and cytokine response was compared using ELISAs while macrophage phenotypes were determined by flow cytometry. MB49 cells were injected into flanks of C57Bl/6J mice followed by intratumoral injection of WT-BCG and rBCG to compare infiltrating Tcells and Teffector responses by flow cytometry. An in vitro trained immunity model of primary human monocytes was used to evaluate epigenetic changes (H3K4Me3) on TNF-a and IL-6 gene promoters by ChIP-PCR. Compared to WT-BCG, intravesical installation of rBCG in MNU-rats resulted in significantly lower tumor involvement and a more potent induction of Th1 cytokines. rBCG treated primary macrophages showed increased IFN I, IL-6, TNF‑a MCP-1 and IL-12 levels concomitant with increased M1 polarization shift. More pronounced IL-6, TNF‑a and IL-1b levels were observed in several human and rodent urothelial carcinoma cells in response to rBCG. Intratumoral injection of rBCG caused increased tumor regression and stronger infiltration of IFN‑g producing CD4+ T cells in MB49 tumors. rBCG elicited more potent epigenetic changes (H3K4Me3) on the TNF-a and IL-6 gene promoters following monocyte training. Increased proinflammatory cytokines, potentiated IFN-g+CD4+ T cells, and increased macrophage reprogramming (increased M1 shift and increased epigenetic changes) contributes to enhanced antitumor efficacy of rBCG over WT-BCG in NMIBC tumor models. Recombinant rBCG may be a novel intravesical immunotherapy for patients with NMIBC.

Oncolytic adenovirus programmed by synthetic gene circuit for cancer immunotherapy

Improving efficacy of oncolytic virotherapy remains challenging due to difficulty increasing specificity and immune responses against cancer and limited understanding of its population dynamics. Here, we construct programmable and modular synthetic gene circuits to control adenoviral replication and release of immune effectors selectively in hepatocellular carcinoma cells in response to multiple promoter and microRNA inputs. By performing mouse model experiments and computational simulations, we find that replicable adenovirus has a superior tumor-killing efficacy than non-replicable adenovirus. We observe a synergistic effect on promoting local lymphocyte cytotoxicity and systematic vaccination in immunocompetent mouse models by combining tumor lysis and secretion of immunomodulators. Furthermore, our computational simulations show that oncolytic virus which encodes immunomodulators can exert a more robust therapeutic efficacy than combinatorial treatment with oncolytic virus and immune effector. Our results provide an effective strategy to engineer oncolytic adenovirus, which may lead to innovative immunotherapies for a variety of cancers.

Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy

Photodynamic Therapy (PDT) with methyl-aminolevulinate acid (MAL-PDT) is being used for the treatment of Basal cell carcinoma (BCC), but recurrences have been reported. In this work, we have evaluated resistance mechanisms to MAL-PDT developed by three BCC cell lines (ASZ, BSZ and CSZ), derived from mice on a ptch+/− background and with or without p53 expression, subjected to 10 cycles of PDT (10thG). The resistant populations showed mesenchymal-like structure and diminished proliferative capacity and size compared to the parental (P) cells. The resistance was dependent on the production of the endogenous photosensitiser protoporphyrin IX in the CSZ cell line and on its cellular localisation in ASZ and BSZ cells. Moreover, resistant cells expressing the p53 gene presented lower proliferation rate and increased expression levels of N-cadherin and Gsk3β (a component of the Wnt/β-catenin pathway) than P cells. In contrast, 10thG cells lacking the p53 gene showed lower levels of expression of Gsk3β in the cytoplasm and of E-cadherin and β-catenin in the membrane. In addition, resistant cells presented higher tumorigenic ability in immunosuppressed mice. Altogether, these results shed light on resistance mechanisms of BCC to PDT and may help to improve the use of this therapeutic approach.

Abstract 5754: Hypoxia-induced tumor plasticity and immune resistance involves an alteration of target recognition by a mechanism involving TGF-beta signaling

Abstract Backgound: Hypoxia is known to shape the tumor microenvironment of developing tumors and contributes to both tumor progression and escape to immune surveillance. Previous work using non-small cell lung carcinoma cells (NSCLC) showed that hypoxia promotes epithelial-mesenchymal transition (EMT) in a fraction of carcinoma cells and that acquisition of a more mesenchymal (Mes) phenotype by certain cancer subclones correlates with an increased propensity to resist cytotoxic T lymphocytes (CTL) attacks as compared to more Epithelial (Epi) cancer subclones. Moreover, numerous studies identified alterations or loss of major histocompatibility complex (MHC) class I as a potential mechanism of tumor escape in cancer. Here, we asked whether hypoxia-induced EMT could regulate MHC class I expression and whether that could impact on resistance to CTL-mediated killing. Methods and Results : By comparing CMH class I in various NSCLC cancer subclones that have experienced hypoxic stress, we found that cancer clones with enhanced Mes features have a reduced expression of CMH class I molecules, as compared to those with a more Epi phenotype. Transient siRNA knockdown of hypoxia effectors HIF-1α or HIF-2α did not affect the expression of CMH class I molecules suggesting that EMT effectors, rather that HIFs, directly regulate CMH class I expression. Among EMT effectors, TGF-β expression was found to be highly upregulated in Mes cancer clones. Moreover, treatment of these cells with TGF-βR inhibitors markedly upregulated CMH class I expression. and as target cells, increases their susceptibility to CTL-mediated killing. However, blocking of TGF-β using Ab to TGF-β during the coculture time had no significant effects on CTL-mediated killing. This suggests that the observed changes in target cell susceptibility occurs through TGF-β-mediated repression of MHC class I rather than by cancer cell production of TGF-β affecting CTL effector functions. Conclusions: These results suggest that the increase in TGF-β signaling, especially found in EMTed Mes carcinoma cells in response to hypoxic stress, contributes to downregulate CMH class I expression in these cells while coinciding with a reduced susceptibilty to CTL attacks. The development of agents targeting hypoxia or EMT components should provide new therapeutic opportunities in combination with current approaches. Citation Format: Stephane Terry, Stephanie Buart, Jean-Paul Thiery, Fathia Mami-Chouaib, Salem Chouaib. Hypoxia-induced tumor plasticity and immune resistance involves an alteration of target recognition by a mechanism involving TGF-beta signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5754.

Comparative analysis of a two-dimensional and three dimensional model of BT-20 triple-negative breast carcinoma cells in response to an antiproliferative agent

Comparative analysis of a two-dimensional and three dimensional model of BT-20 triple-negative breast carcinoma cells in response to an antiproliferative agent

Inhibition of Protein Phosphatase 2A Sensitizes Mucoepidermoid Carcinoma to Chemotherapy via the PI3K-AKT Pathway in Response to Insulin Stimulus

Background/Aims: Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that mediates cell cycle regulation and metabolism. Mounting evidence has indicated that PP2A inhibition exhibits considerable anticancer potency in multiple types of human cancers. However, the efficacy of PP2A inhibition remains unexplored in mucoepidermoid carcinoma (MEC), especially in locally advanced and metastatic cases with limited systemic treatment. In this study, we demonstrated the therapeutic potency of LB100 in mucoepidermoid carcinoma. Methods: In this study, the expression of PP2A was evaluated using immunohistochemical (IHC) staining. The effects associated with LB100 alone and in combination with cisplatin for the treatment of mucoepidermoid carcinoma were investigated both in vitro, regarding metabolism, proliferation, and migration, and in vivo in a mucoepidermoid carcinoma xenograft model. In addition, with LB100 treatment and in response to an insulin stimulus, the expression levels and phosphorylation levels of targets in the PI3K-AKT pathway were determined using western blot analysis and immunoblotting. Results: The expression of protein phosphatase 2A was significantly upregulated in the clinical specimens of high-grade MECs compared with those of low-/medium-grade MECs and normal controls. In this article, we report that a small molecule PP2A inhibitor, LB100, decreased cellular viability and glycolytic activity and induced G2/M cell cycle arrest. Importantly, LB100 enhanced the efficacy of cisplatin in mucoepidermoid carcinoma cells both in vitro and in vivo. PP2A inhibition by LB100 increased the phosphorylation of insulin receptor substrate 1(IRS-1) on serine residues, downregulated the expression of phosphatidylinositol 3-kinase (PI3K) p110 alpha subunit and dephosphorylated AKT at Ser473 and Thr308 in mucoepidermoid carcinoma cells in response to insulin stimulus. Conclusion: These results highlight the translational potential of PP2A inhibition to synergize with cisplatin in mucoepidermoid carcinoma treatment.

Collagen Matrix Density Drives the Metabolic Shift in Breast Cancer Cells

Increased breast density attributed to collagen I deposition is associated with a 4–6 fold increased risk of developing breast cancer. Here, we assessed cellular metabolic reprogramming of mammary carcinoma cells in response to increased collagen matrix density using an in vitro 3D model. Our initial observations demonstrated changes in functional metabolism in both normal mammary epithelial cells and mammary carcinoma cells in response to changes in matrix density. Further, mammary carcinoma cells grown in high density collagen matrices displayed decreased oxygen consumption and glucose metabolism via the tricarboxylic acid (TCA) cycle compared to cells cultured in low density matrices. Despite decreased glucose entry into the TCA cycle, levels of glucose uptake, cell viability, and ROS were not different between high and low density matrices. Interestingly, under high density conditions the contribution of glutamine as a fuel source to drive the TCA cycle was significantly enhanced. These alterations in functional metabolism mirrored significant changes in the expression of metabolic genes involved in glycolysis, oxidative phosphorylation, and the serine synthesis pathway. This study highlights the broad importance of the collagen microenvironment to cellular expression profiles, and shows that changes in density of the collagen microenvironment can modulate metabolic shifts of cancer cells.

Influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin

To explore the influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin. Transfected nasopharyngeal carcinoma 5-8F cell lines with pCDH-CMV-PinX1-copGFP vector constructed by lentivirus to generate Lenti-PinX1-5-8F cells containing PinX1 gene, using Lenti-Ctrl-5-8F cell (blank vector without PinX1 gene was used to transfect 5-8F cell lines) and 5-8F cell as controls. Expression of PinX1 gene, telomerase activity, the inhibition of cancer cells proliferation, combined anticancer effect with Cisplatin and the expression of lung resistance protein (LRP) and Bcl-2 were detected with fluorescent quantitation polymerase chain reaction (PCR), flow cytometry, thiazolyl blue (MTT) method, areole test, Western blot and drug sensitivity test, respectively, in four groups (Lenti-PinX1-5-8F cell + Cisplatin, Lenti-PinX1-5-8F cell, Cisplatin and 5-8F cell) so as to explore the influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin. The telomerase activity in Lenti-PinX1-5-8F cell (0.146 ± 0.004) was lower than those in the other two control cells (Lenti-Ctrl-5-8F cell: 0.967 ± 0.016, 5-8F cell: 1.000 ± 0.034, both P < 0.01). The cancer cell biological activity could be intensively inhibited by 16 µg/ml Cisplatin after lower level telomerase activity induced by PinX1 gene. Proliferation index (PI) (%) in Lenti-PinX1-5-8F cell + Cisplatin (14.39 ± 3.66) was also less than the other groups (Lenti-PinX1-5-8F cell, Cisplatin and 5-8F cell groups, 32.97 ± 3.00, 31.18 ± 4.24 and 47.19 ± 4.19, all P < 0.01). And same time, the expressions of LRP (0.64 ± 0.14) and Bcl-2 (0.57 ± 0.12) protein in Lenti-PinX1-5-8F cells were obviously reduced than those in other two group cells (Lenti-Ctrl-5-8F cell: 0.84 ± 0.19 and 0.81 ± 0.16; 5-8F cell: 0.83 ± 0.35 and 0.78 ± 0.27; all P < 0.01). PinX1 gene can enhance the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin, which may be mediated by the down-regulation of telomerase activity and the inhibition of LRP and Bcl-2 gene in nasopharyngeal carcinoma cells.

The surfactant dipalmitoylphophatidylcholine modifies acute responses in alveolar carcinoma cells in response to low-dose silver nanoparticle exposure.

Nanotechnology is a rapidly growing field with silver nanoparticles (AgNP) in particular utilized in a wide variety of consumer products. This has presented a number of concerns relating to exposure and the associated toxicity to humans and the environment. As inhalation is the most common exposure route, this study investigates the potential toxicity of AgNP to A549 alveolar epithelial carcinoma cells and the influence of a major component of lung surfactant dipalmitoylphosphatidylcholine (DPPC) on toxicity. It was illustrated that exposure to AgNP generated low levels of oxidative stress and a reduction in cell viability. While DPPC produced no significant effect on viability studies its presence resulted in increased reactive oxygen species formation. DPPC also significantly modified the inflammatory response generated by AgNP exposure. These findings suggest a possible interaction between AgNP and DPPC causing particles to become more reactive, thus increasing oxidative insult and inflammatory response within A549 cells.

Simultaneous irradiation of fibroblasts and carcinoma cells repress the secretion of soluble factors able to stimulate carcinoma cell migration.

Stroma mediated wound healing signals may share similarities with the ones produced by tumor’s microenvironment and their modulation may impact tumor response to the various anti-cancer treatments including radiation therapy. Therefore we conducted this study, to assess the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts irrespective of their RhoB status do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms which are TGF-β1 and MMP-mediated respectively. Lastly, we found that simultaneous irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This last result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response.

Inhibition of Mus81 by siRNA enhances sensitivity to 5-FU in breast carcinoma cell lines

PurposeOne of the most challenging aspects of breast carcinoma chemotherapy is the rapid acquirement of drug resistance. Improving the sensitivity to chemotherapeutic drugs is very important for successful treatment. Mus81 plays an important role in maintaining the stability of the genome and DNA repair. However, recent studies suggested that Mus81 expression levels correlate well with resistance to DNA-damaging drugs. The present study aimed to investigate the role of Mus81 on the chemosensitivity of breast carcinoma cells in response to 5-fluorouracil (5-FU), a chemotherapeutic drug that is widely used for treatment of breast malignancies.MethodsThe expression of Mus81 in MCF-7 and T47D cells was suppressed by small interfering RNA (siRNA). mRNA and protein levels of Mus81 were analyzed by quantitative real-time polymerase chain reaction and Western blot. Cell viability and colony survival were determined by Cell Counting Kit-8 and plate colony formation assay, respectively. Cell cycle and apoptosis were detected by flow cytometry.Results5-FU inhibited the cell viability of MCF-7 and T47D cells in a concentration-dependent manner. We found that the Mus81-silenced MCF-7 and T47D cells exhibited decreased cell viability and clonogenic survival, but increased G2 accumulation, in response to 5-FU. In addition, Mus81 deficiency resulted in increased apoptosis and p53 expression in MCF-7 after 5-FU treatment. However, Mus81 deficiency did not affect the apoptosis of T47D cells with 5-FU.ConclusionTaken together, our data suggest that Mus81 inhibition significantly increased the chemosensitivity of MCF-7 and T47D cells in response to 5-FU. Thus, Mus81 siRNA is potentially a useful adjuvant strategy for breast cancer chemotherapy.

Abstract A26: Colon cancer prevention through combinations of dietary or pharmacological agents

Abstract Our studies aim at developing combinations of dietary or pharmacological agents that lower the risk for colorectal cancer (CRC). In vitrostudies have demonstrated that histone deacetylase inhibitors (HDACis) induce programmed death (apoptosis) in CRC cells with mutations in the WNT/beta-catenin pathway, and the apoptotic levels depend upon the hyperactivation of WNT signaling. Since mutations in the WNT/catenin pathway are present in almost 80% of all sporadic CRCs, dietary and synthetic HDACis are promising anti-CRC agents. However, HDACi-initiated apoptosis of CRC cells is counteracted by induction of compensatory proliferation supported by the AKT and JAK/STAT cell survival pathways. Therefore, we ascertained whether agents that suppress cell survival signaling augment the apoptotic effect of HDACis. We utilized apoptotic assays and immunoblotting analyses to ascertain the ability of compensatory pathway inhibitors to augment the apoptosis induced by dietary and pharmacological HDACis in colorectal neoplastic cells. The effects of the agents were assessed in HDACi-resistant colorectal cancer (HCT-R) and early colonic adenoma (LT97) cells. A combination treatment of CRC cells with a synthetic HDACi (LBH589), an AKT inhibitor (MK2206), and a JAK/STAT inhibitor (pyridone 6) validated the concept that the apoptotic effect of a HDACi is augmented by inhibitors of compensatory proliferation. Such pharmacological approach to CRC prevention might be applicable for populations at high risk for colon cancer (e.g., CRC patients with partial colonic resection, chronic inflammatory bowel disease, history of colonic polyps). Interestingly, diet-derived modulators of compensatory survival pathways affect differently HDACi-induced apoptosis in colonic carcinoma and early adenoma cells. Thus, caffeic acid phenethyl ester (CAPE, an AKT inhibitor contained in some honeybee propolis), and gallotannin (a JAK/STAT inhibitor derived from some fruit and wine) augment the apoptotic effect of the dietary HDACi butyrate in colonic carcinoma HCT-R cells, but not in adenoma LT97 cells. Resveratrol and pterostilbene (phenols found in grapes and blueberries) both suppress butyrate-induced apoptosis in colorectal carcinoma HCT-R cells, and augment butyrate-induced apoptosis in early adenoma cells in the presence of CAPE. Since at physiologically achievable concentrations CAPE, gallotannin, resveratrol, and pterostilbene alone or in combination do not induce apoptosis, the signaling pathways targeted by these compounds in butyrate-treated colonic neoplastic cells are true apoptosis-induced compensatory proliferation pathways. Our data suggest that different compensatory proliferation pathways are activated in colonic adenoma and carcinoma cells in response to the diet-derived HDACi butyrate. Further analyses should ascertain the identity of these cell survival signaling pathways. In conclusion, the rationale of combining inducers of apoptosis (e.g., HDACis) with inhibitors of compensatory proliferation may result in the design of supplements with a powerful CRC-protective effect. The next step in the development of such dietary supplements is ascertaining their effect in animal models of intestinal cancer. Citation Format: Eric Drago, Michael Bordonaro, Jeffrey Marteslo, Patricia Melvin, Wafa Atamna, Darina Lazarova. Colon cancer prevention through combinations of dietary or pharmacological agents. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A26.

Contributors to HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guérin

Our group previously noted that HMGB1 release by urothelial carcinoma cells occurs as a consequence of bacillus Calmette-Guérin induced nonapoptotic cell death. Additional studies demonstrated that HMGB1 release in response to bacillus Calmette-Guérin is required for the invivo tumor response to bacillus Calmette-Guérin. We evaluated the steps required for HMGB1 release by human urothelial carcinoma cells in response to bacillus Calmette-Guérin exposure. We used the T24 and 253J human urothelial carcinoma cell lines. HMGB1 concentrations in cell culture supernatant with and without bacillus Calmette-Guérin treatment served as the principal end point to assess the role of potentially involved variables. Specific techniques were used to determine the role of α5β1 antigen receptor cross-linking, TLR signaling, inducible nitric oxide synthase expression/nitric oxide production, p21 expression, and bacillus Calmette-Guérin adherence, internalization and viability. Cross-linking of α5β1 integrin or signaling through TLR2/4 did not contribute to HMGB1 release. Optimal HMGB1 release required bacillus Calmette-Guérin adherence and internalization. Bacillus Calmette-Guérin viability correlated with the magnitude of HMGB1 release. Inhibition of oxidative stress and p21 expression in response to bacillus Calmette-Guérin decreased the magnitude of HMGB1 release. Bacillus Calmette-Guérin induced nonapoptotic cell death and HMGB1 release occur as a consequence of a complex multistep process. Understanding the steps and mechanisms involved in the induced HMGB1 release would provide an opportunity for targeted strategies to improve bacillus Calmette-Guérin treatment efficacy.

HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guérin Functions as a Paracrine Factor to Potentiate the Direct Cellular Effects of Bacillus Calmette-Guérin

Prior study demonstrated that HMGB1 release by urothelial carcinoma cells in response to bacillus Calmette-Guérin is required for an in vivo antitumor effect. We evaluated the direct effects of HMGB1 on the in vitro response of urothelial carcinoma cells to bacillus Calmette-Guérin. Two human urothelial carcinoma cell lines were used to study the effect of exogenous HMGB1 alone and combined with bacillus Calmette-Guérin on the tumor cell response to bacillus Calmette-Guérin. Antibody mediated blockade of receptors for HMGB1 or HMGB1 protein was used to determine the contribution of paracrine HMGB1 release to bacillus Calmette-Guérin biological effects. Response end points evaluated included the activation of intracellular signaling pathways, gene transactivation and cytotoxicity. Urothelial carcinoma cells expressed the receptor for HMGB1 signaling. Antibody blockade of the RAGE receptor confirmed the dependence of signaling in response to HMGB1 on RAGE function. Exogenous HMGB1 activated cell signaling pathways for NFκB, NRF2 and CEBP. Quantitative reverse transcriptase-polymerase chain reaction on a panel of bacillus Calmette-Guérin responsive genes revealed peak expression resulting from the combination of bacillus Calmette-Guérin and HMGB1. Blockade of paracrine HMGB1 released in response to bacillus Calmette-Guérin using HMGB1 and/or RAGE receptor blocking antibodies showed a significant decrease in gene expression relative to that of bacillus Calmette-Guérin alone. HMGB1 potentiated the cytotoxic effects of bacillus Calmette-Guérin. HMGB1 released by urothelial carcinoma cells after bacillus Calmette-Guérin treatment functions as a paracrine factor to potentiate the urothelial carcinoma cell response to bacillus Calmette-Guérin. This paracrine activity likely contributes to the dependence of an in vivo tumor response on HMGB1 release.

Autophagy, cell death and sustained senescence arrest in B16/F10 melanoma cells and HCT-116 colon carcinoma cells in response to the novel microtubule poison, JG-03-14

Previous studies have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to kill breast tumor cells primarily through the promotion of autophagy. The current work was designed to determine whether autophagy was, in fact, the primary mode of action as well as susceptibility to JG-03-14 in two additional tumor cell models, the B16/F10 murine melanoma cell line and the HCT-116 human colon cancer cell line. Drug cytotoxicity was monitored based on viable cell number and clonogenic survival. Apoptosis was assessed by DAPI staining, the TUNEL assay and/or FACS analysis. Autophagy was monitored based on staining with acridine orange, redistribution and punctuation of RFP-LC3 and electron microscopy as well as p62 degradation. Senescence was evaluated based on β-galactosidase staining and alterations in cell morphology. Drug effects were also evaluated in a murine model of B16/F10 cells that localizes to the lungs while peripheral neuropathy was assessed by three complementary behavioral assays. Both HCT-116 colon cancer cells and B16/F10 melanoma cells were sensitive to JG-03-14 in that the drug demonstrated tumor cell killing. However, there was minimal induction of apoptosis. In contrast, there was clear evidence for autophagy and autophagic flux while the residual surviving cells appeared to be in a state of irreversible senescence. Inhibition of drug-induced autophagy in either the melanoma cells or the colon carcinoma cells was only slightly protective as the cells instead died by apoptosis. JG-03-14 reduced the size of tumor nodules in mice lungs; furthermore, the drug did not promote peripheral neuropathy. Taken together with evidence for its actions as a vascular disrupting agent, these observations support the potential utility of JG-03-14 to effectively treat malignancies that might be resistant to conventional chemotherapy through evasion of apoptosis.

Abstract 2344: Distinct combinations of signaling pathways promote migration of MDA-MB-231 breast carcinoma cells in response to different stimuli

Abstract Cancer cell invasion and metastasis are predicated on the activation of signaling pathways that control dynamic organization of cytoskeletal architecture. The coordinate reorganization of an actin network pushing at the leading edge and an actin-myosin mediated retraction at the rear of the cell facilitates cell polarity to allow directed movement. Motility of the highly invasive breast carcinoma cell line MDA-MB-231 is promoted by various stimuli including lysophosphatidic acid (LPA), hepatocyte growth factor (HGF) and epidermal growth factor (EGF). The goal of this project is to delineate the signaling pathways mediating motility in response to each of these stimuli. Identifying common and conditional pathways mediating cell motility has important implications for therapeutic intervention of cancer cell invasion and metastasis. We conducted migration, invasion, and morphology assays in the presence of inhibitors for pathways known to contribute to the cytoskeletal reorganization required for cell migration. LPA, HGF and EGF were evaluated for their capacity to signal through the Rho family small GTPases, Rho and Rac, the MAP kinase pathway via MEK1/2 and myosin light chain kinase (MLCK) to influence cell migration and invasion. LPA is a recognized component of breast cancer progression and potent stimulator of Rho activity, which has been extensively implicated in the migration of carcinoma cells in a variety of cancers including breast. Surprisingly, LPA-stimulated migration of MDA-MB-231 cells was not affected by Rho inhibitors but was instead sensitive to Rac inhibition. In contrast, Rho was required for HGF and EGF-stimulated migration of MDA-MB-231 cells while Rac had no effect on these processes. Other common pathways mediating cell motility including MLCK are used by all three chemoattractants. MDA-MB-231 cells employed all of the pathways examined for invasion, potentially reflecting the complexity of navigating in a three dimensional environment. These observations suggest that while multiple signaling pathways contribute to the migratory potential of a cancer cell, not all pathways are operating under all conditions. Different context-specific pathways may supplement a core of common pathways to create a functional signal but the characteristics of the response will likely rely on the particular combination used by the cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2344. doi:10.1158/1538-7445.AM2011-2344