Preventing Tumor Cell Death Research Articles

RNF31 promotes tumorigenesis via inhibiting RIPK1 kinase-dependent apoptosis.

It is well established that interferon (IFN) and tumor necrosis factor (TNF) could synergistically promote antitumor toxicity and avoid resistance of antigen-negative tumors during cancer immunotherapy. The linear ubiquitin chain assembly complex (LUBAC) has been widely known to regulate receptor-interacting protein kinase-1(RIPK1) kinase activity and TNF-mediated cell death during inflammation and embryogenesis. However, whether LUBAC and RIPK1 kinase activity in tumor microenvironment could regulate antitumor immunity are still not very clear. Here, we demonstrated a cancer cell-intrinsic role of LUBAC complex in tumor microenvironment to promote tumorigenesis. Lacking LUBAC component RNF31 in B16 melanoma cells but not immune cells including macrophages or dendritic cells greatly impaired tumor growth by increasing intratumoral CD8+ T cells infiltration. Mechanistically, we found that tumor cells without RNF31 shown severe apoptosis-mediated cell death caused by TNFα/IFNγ in the tumor microenvironment. Most importantly, we found that RNF31 could limit RIPK1 kinase activity and further prevent tumor cell death in a transcription-independent manner, suggesting a crucial role of RIPK1 kinase activity in tumorigenesis. Together, our results demonstrate an essential role of RNF31 and RIPK1 kinase activity in tumorigenesis and imply that RNF31 inhibition could be harnessed to enhance antitumor toxicity during tumor immunotherapy.

Targeting Autophagy in Thyroid Cancer: EMT, Apoptosis, and Cancer Stem Cells.

Autophagy is a highly conserved recycling process through which cellular homeostasis is achieved and maintained. With respect to cancer biology, autophagy acts as a double-edged sword supporting tumor cells during times of metabolic and therapeutic stress, while also inhibiting tumor development by promoting genomic stability. Accumulating evidence suggests that autophagy plays a role in thyroid cancer, acting to promote tumor cell viability and metastatic disease through maintenance of cancer stem cells (CSCs), supporting epithelial-to-mesenchymal transition (EMT), and preventing tumor cell death. Intriguingly, well-differentiated thyroid cancer is more prevalent in women as compared to men, though the underlying molecular biology driving this disparity has not yet been elucidated. Several studies have demonstrated that autophagy inhibitors may augment the anti-cancer effects of known thyroid cancer therapies. Autophagy modulation has become an attractive target for improving outcomes in thyroid cancer. This review aims to provide a comprehensive picture of the current knowledge regarding the role of autophagy in thyroid cancer, focusing on the potential mechanism(s) through which inhibition of autophagy may enhance cancer therapy and outcomes.

IL-7 Either Promotes or Inhibits Autophagy, Depending on the Stress Context, to Promote the Viability of T-Cell Acute Lymphoblastic Leukemia Cells

T-cell acute lymphoblastic leukemia (T-ALL), an aggressive and common childhood hematological malignancy, arises from clonal expansion of T-cell progenitors. Autophagy is a homeostatic process characterized by the sequestration of cytoplasmic compartments within double-membrane vesicles (autophagosomes) to promote their degradation. Importantly, autophagy is upregulated during starvation and cellular stress, as a compensatory mechanism to provide nutrients and stress relief. By mitigating stress and allowing cell survival, autophagy may serve as a pro-tumoral mechanism. On the other hand, persistent autophagy can lead to cell death and thereby prevent tumor growth. Interleukin-7 (IL-7), a cytokine produced by the bone marrow and thymic stroma, is essential for normal T-cell development. However, IL-7-mediated signaling can also contribute to leukemogenesis. A majority of T-ALL patients (~70%) expresses the IL-7 receptor and IL-7 accelerates T-ALL progression in vivo and promotes T-ALL cell proliferation, survival and metabolic activation in vitro via PI3K/Akt/mTOR pathway (a master negative regulator of autophagy). IL-7 can also activate MEK/Erk pathway (which has been implicated in promotion of autophagy). Because IL-7 has the ability to activate signaling pathways with potentially opposing roles in autophagy regulation, we explored whether IL-7 impacted on the autophagic process in T-ALL cells and sought to elucidate the molecular mechanisms and functional consequences of IL-7-mediated autophagy regulation under different culture conditions: a) medium with 10% serum - a scenario where cells have optimal growth conditions, b) medium with low (1%) serum - to mimic a milieu with nutrient stress (which may happen in densely populated leukemic niches in vivo), and c) regular medium plus asparaginase (ASNase) - to induce treatment-imposed stress.Using IL-7-responsive T-ALL cell lines and patient-derived xenograft (PDX) samples, we show that, in optimal culture conditions, IL-7 leads to a decrease in LC3-I/-II conversion and a reduction in both LC3 puncta and autophagosome/autolysosome formation, as determined by immunoblot, confocal microscopy, flow cytometry and electron microscopy - indicating that IL-7 inhibits autophagy in T-ALL. Using signaling-specific small molecule inhibitors (UO126 for MEK/Erk; LY294002 and rapamycin for PI3K/Akt/mTOR) we found that IL-7-mediated regulation of autophagy occurs in a complex manner that involves concomitant triggering of both pro- (via MEK/Erk) and anti- (via PI3K/Akt/mTOR) autophagic signaling, with the effects of the latter prevailing over the former. In this scenario, IL-7-mediated viability relies on PI3K/Akt/mTOR pathway and, as expected, autophagy inhibition (using MRT68921) does not prevent the ability of IL-7 to promote leukemia cell survival. In contrast, under serum starvation IL-7 promotes autophagy, and IL-7-mediated leukemia cell viability partially relies on autophagy activation, and strictly requires MEK/Erk activation. Mechanistically, we provide evidence that depending on the culture conditions, IL-7 can balance the relative activation of PI3K/Akt/mTOR and MEK/Erk pathways, inhibiting or facilitating autophagy, in order to consistently promote T-ALL cell viability. We further extended our studies to a therapy-related scenario and found that under ASNase treatment, IL-7 still promotes increased survival of leukemic cells, a possible mechanism of treatment resistance. Functionally, we demonstrate that the IL-7-mediated increase in survival under ASNase treatment is, in part, mediated via activation of autophagy, suggesting that combining ASNase administration with autophagy inhibitors may be an attractive strategy to prevent resistance.In summary, our results indicate that IL-7 makes use of a ‘flexible strategy’ to promote T-ALL cell viability by activating both pro- and anti-autophagic pathways, which are differentially recruited, depending on the microenvironmental conditions, to prevent tumor cell death. Moreover, our findings strengthen the notion that combination therapies against PI3K/Akt/mTOR and MEK/Erk pathways may constitute a valid therapeutic avenue and highlight the potential of using autophagy inhibitors to prevent microenvironment-induced chemotherapy resistance in T-ALL. DisclosuresBarata:Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents.

Bcl-2 degradation is an additional pro-apoptotic effect of polo-like kinase inhibition in cholangiocarcinoma cells.

AIMTo examine the influence on apoptotic mechanisms following inhibition of polo-like kinases as therapeutically approach for cholangiocellular cancer treatment.METHODSAs most cholangiocarcinomas are chemotherapy-resistant due to mechanisms preventing tumor cell death, we investigated the effect of Cisplatin on cholangiocellular carcinoma (CCA) cell lines KMCH-1 and Mz-Ch-1. Polo-like kinases (PLK) are important regulators of the cell cycle and their inhibition is discussed as a potential therapy while PLK inhibition can regulate apoptotic mediators. Here, cells were treated with PLK inhibitor BI6727 (Volasertib), Cisplatin, and in combination of both compounds. Cell viability was assessed by MTT; apoptosis was measured by DAPI staining and caspase-3/-7 assay. Western blot and qRT-PCR were used to measure expression levels of apoptosis-related molecules Bax and Bcl-2.RESULTSThe cell viability in the CCA cell lines KMCH-1 and Mz-Ch-1 was reduced in all treatment conditions compared to vehicle-treated cells. Co-treatment with BI6727 and cisplatin could even enhance the cytotoxic effect of cisplatin single treatment. Thus, co-treatment of cisplatin with BI6727 could slightly enhance the cytotoxic effect of the cisplatin in both cell lines whereas there was evidence of increased apoptosis induction solely in Mz-Ch-1 as compared to KMCH-1. Moreover, PLK inhibition decreases protein levels of Bcl-2; an effect that can be reversed by the proteasomal degradation inhibitor MG-132. In contrast, protein levels of Bax were not found to be altered by PLK inhibition. These findings indicate that cytotoxic effects of Cisplatin in Mz-Ch-1 cells can be enhanced by cotreatment with BI6727.CONCLUSIONIn conclusion, BI6727 treatment can sensitize CCA cells to cisplatin-induced apoptosis with proteasomal Bcl-2 degradation as an additional pro-apoptotic effect.

Gonadotropin-mediated chemoresistance: Delineation of molecular pathways and targets.

BackgroundHuman chorionic gonadotropin (hCG) has essential roles in pregnancy. Reports linking hCG in non-trophoblastic tumors with poor patient prognosis has spurred interest in patho-physiological roles the hormone might play.MethodsThe ability of hCG to prevent tumor cell death and sustain viability in the presence of chemotherapeutic drugs was assessed and potential synergies with TLR ligands explored. hCG-induced up-modulation of genes involved in chemoresistance was documented and targets validated by siRNA knock-down. Whether hCG could drive collaboration between tumor cells and macrophages in the production of IL-6 and consequent chemoresistance was assessed. The effects of concurrent anti-hCG immunization and chemotherapy on the growth of syngeneic murine tumors were evaluated.ResultshCG maintained basal levels of cytokine secretion by tumor cells exposed to chemotherapeutic drugs, and enhanced viability and proliferation; pre-treatment with hCG also decreased apoptosis, as assessed by Annexin-V binding and the cleavage of caspase 3. While co-incubation with hCG along with several TLR ligands mediated heightened chemo-resistance, TLR-2/6 and TLR-9 ligands increased the phosphorylation of JNK, and TLR-2 and TLR-8 ligands the phosphorylation of ERK in presence of hCG and curcumin, providing evidence of tri-molecular synergy. The hormone increased the transcription and/or expression of molecular intermediates (SURVIVIN, HIF-1α, PARP-1, Bcl-2, c-FLIP, KLK-10, XIAP, c-IAP-1) associated with chemo-resistance and increased levels of stress modulators (PON2, HO-1, HSP27 and NRF-2). siRNAs to SURVIVIN, NRF-2, HO-1 and HIF-1α attenuated hCG-mediated chemo-resistance. hCG-conditioned tumor cell supernatants induced heightened secretion of IL-6 and TNF-α from peripheral blood adherent cells and secreted IL-6 imparted chemo-resistance to naïve tumor cells. Co-administration of curcumin along with an anti-hCG vaccine (hCGβ conjugated to Tetanus Toxoid (TT)) to mice carrying syngeneic tumors resulted in significantly enhanced benefits on animal survival; synergy was demonstrated between anti-hCG antibodies and curcumin in the reduction of tumor cell viability.ConclusionsThe data suggest that hCG, via direct as well as collaborative effects with TLR ligands and accessory cell-secreted cytokines, mediates chemo-resistance in gonadotropin-sensitive tumors and outlines the potential benefits of combination therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1938-x) contains supplementary material, which is available to authorized users.

Abstract 5473: The sensitivity of targeting genomic BCL2 by PNT2258 is linked to chromosomal rearrangements and proliferative rate of tumor types

Abstract Background: The BCL2-mediated anti-apoptotic phenotype is a contributor to the genesis and maintenance of a broad variety of tumors. BCL2 is also implicated in the regulation of the cell cycle by playing a role in the transition between quiescence and the cycling state. Further, chromosomal rearrangements, including t(14;18), up-regulate BCL2 transcription, preventing tumor cell death in B-cell lymphomas. PNT2258 is a DNA interference (DNAi) therapeutic targeted against BCL2 that is undergoing clinical evaluation in patients with hematological malignancies. PNT2258 contains PNT100, a single-stranded phosphodiester DNA oligodeoxynucleotide, encapsulated in protective liposomes. Material and Methods: Lymphomas cell lines with distinct genetic characteristics, namely, WSU-FSCCL, characterized by t(14;18) BCL2 and t(8;14) CMYC rearrangements, WSU-DLCL2, characterized by t(14;18) BCL2 rearrangement, and WSU-WM, characterized as lacking t(14;18) BCL2, but having t(8;14) CMYC and t(12;17) rearrangements were tested for cell viability at 24, 48, 72, and 96 hours post-exposure to PNT2258 at concentrations of 2.5, 5, and 10 μM. Normalized maximum cell kill at 96 hours was used in each cell line to adjust for differences in growth rates and to calculate sensitivity. Results: Dose-dependent effects were observed across all three cell lines. WSU-FSCCL was the most proliferative cell line, and correspondingly most sensitive to the effects of PNT2258, with a control doubling time of 29.6h and 1% of viable control cells remaining at 96h at 10 μM. The next most sensitive cell line was WSU-DLCL2, having a 60.6h control doubling time and 11% of the viable control cells remaining at 96h at 10 μM. WSU-WM exhibited a control doubling time of 35.5h and 21% of viable control cells remaining at 96h at 10 μM. These findings parallel the single agent activity of PNT2258 against xenograft tumor models containing BCL2 or CMYC chromosomal rearrangements (AACR Meeting Abstracts, Apr 2007; 2007: 4889). Conclusions: The effects of the clinical therapeutic PNT2258 against cell lines with well-characterized growth and genetic drivers were examined. Findings show lymphoma cell lines harboring the t(14;18) rearrangement (WSU-FSCCL and WSU-DLCL2) were most sensitive to PNT2258 and in this context a higher proliferative rate is linked to sensitivity. These data suggest that commonly used clinical assays of proliferation and tumor “aggressiveness,” such as Ki-67 and PET/CT standardized uptake values (SUVs), may be useful in conjunction with cytogenetic analysis (e.g. for t(14;18) and/or t(8,14) by fluorescence in situ hybridization (FISH), to select patients with tumors that may be responsive to the effects of PNT2258. Citation Format: Michael J. Woolliscroft, Abdul-Shukkur Ebrahim, Richard A. Messmann, Shari K. Gaylor, Mina P. Sooch, Ayad Al-Katib, Wendi V. Rodrigueza. The sensitivity of targeting genomic BCL2 by PNT2258 is linked to chromosomal rearrangements and proliferative rate of tumor types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5473. doi:10.1158/1538-7445.AM2014-5473

The mTOR Kinase Inhibitors, CC214-1 and CC214-2, Preferentially Block the Growth of EGFRvIII-Activated Glioblastomas

mTOR pathway hyperactivation occurs in approximately 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here, we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacologic strategy to overcome it. We conducted in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin-resistant signaling and blocking glioblastoma growth and a novel single-cell technology-DNA Encoded Antibody Libraries-was used to identify mechanisms of resistance. Here, we show that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling, block mTORC2 signaling, and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes glioblastoma cells and orthotopic xenografts to CC214-1- and CC214-2-induced cell death. These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in glioblastoma, and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. In addition, this study also shows a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacologic strategy for overcoming it.

Abstract 3529: Effect of PNT2258, an anti-BCL2 DNA-interference drug, on tumor growth and immunological markers in mice and humans.

Abstract Background: The dysregulation of apoptosis is a defining characteristic of malignant cells where excessive concentration of BCL2 protein contributes to the anti-apoptotic phenotype, driving development and subsequent resistance to therapy. Chromosomal translocations, including the t(14;18) rearrangement, up-regulate BCL2 transcription, preventing tumor cell death in B-cell lymphomas. A new class of therapeutic agents, called DNA interference (DNAi) drugs, exert their therapeutic effect by selectively blocking the transcription of oncogenes. PNT2258 is the lead DNAi drug currently undergoing clinical evaluation in patients with hematological malignancies. PNT2258 contains PNT100, a single stranded, native sequence oligonucleotide targeted against BCL2, which is delivered to cancer cells in a protective liposomal transport system. We present data on the anti-tumor and immunomodulatory effects of PNT228 in mice compared with that observed in patients with advanced solid tumors treated with escalating doses of PNT2258. Material and Methods: Twenty-two patients received PNT2258 doses ranging from 1 to 150 mg/mˆ2 as part of a Phase I dose-escalation study in patients with advanced, treatment refractory solid tumors. Patient plasma specimens were analyzed using a 61-marker multiplex immunoassay. Balb/c and WSU-DLCL2 xenograft mice received PNT2258 or an encapsulated scrambled control (oligonucleotide) sequence at a dose of 20 mg/kg by intravenous infusion. Murine plasma was analyzed with a 37-marker multiplex immunoassay. Results: In the murine xenograft model, PNT2258 demonstrated sequence-specific anti-tumor and anti-BCL2 activity not observed with the scrambled control. Xenograft mice demonstrated a strong innate immune response that was very similar in magnitude for both PNT2258 and scrambled control. Balb/c mice exhibited a broad and relatively weak immune response to PNT2258 and a stronger response to the scrambled control suggesting activation of both adaptive and innate immune responses. In patients, PNT2258 did not produce clinical signs of immune stimulation. Multiplex immunoassay revealed a lack of significant drug-induced modulation of inflammatory biomarkers following treatment. PNT2258 did induce statistically significant dose-dependent changes in IP-10, leptin, MIP-1β, MCP-1, IL-17F, and IL-1RA consistent with a mechanistic response to BCL2 suppression. Conclusions: Biomarker response profiles of encapsulated human sequence-specific oligonucleotide therapeutics in mice are not predictive of responses in humans. PNT2258 is safe and well tolerated in patients with no evidence of an innate (TLR) response. The observed biomarker profile provides mechanistic confirmation of drug-induced BCL2 suppression at all doses tested. Leptin represents a unique biomarker that may be used to monitor PNT2258 anti-BCL2 activity in the clinic. Citation Format: Elzbieta Izbicka, Robert Streeper, Michael J. Wick, Drew Rasco, Amita Patnaik, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Shari Gaylor, Michael J. Woolliscroft, Richard A. Messmann, Wendi V. Rodrigueza. Effect of PNT2258, an anti-BCL2 DNA-interference drug, on tumor growth and immunological markers in mice and humans. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3529. doi:10.1158/1538-7445.AM2013-3529

Wnt/β-catenin signaling enhances hypoxia-induced epithelial–mesenchymal transition in hepatocellular carcinoma via crosstalk with hif-1α signaling

Epithelial-mesenchymal transition (EMT) is a critical process for tumor invasion and metastasis. Hypoxia may induce EMT, and upregulated β-catenin expression has been found in various tumors. In this study, we investigate the role of β-catenin in hypoxia-induced EMT in hepatocellular carcinoma (HCC). Induction of EMT in HCC cell lines by hypoxia was confirmed by altered morphology, expression change of EMT-associated markers and enhanced invasion capacity. We showed that hypoxia-induced EMT could be enhanced by addition of recombinant Wnt3a while it was repressed by β-catenin small interfering RNA. An interaction between β-catenin and hypoxia-induced factor-1α (hif-1α) was found, and an underlying competition for β-catenin between hif-1α and T-cell factor-4 was implied. Notably, increased hif-1α activity was accompanied with more significant EMT features. We also showed that the pro-EMT effect of β-catenin in hypoxia was deprived in the absence of hif-1α. Moreover, β-catenin was found to be responsible for the maintenance of viability and proliferation for tumor cells undergoing hypoxia. We further showed a correlation between hif-1α and β-catenin expression, and corresponding expression of EMT-associated markers in human HCC tissues. Our results suggest that Wnt/β-catenin signaling enhances hypoxia-induced EMT in HCC by increasing the EMT-associated activity of hif-1α and preventing tumor cell death.

Hyaluronan-CD44v3 Interaction with Oct4-Sox2-Nanog Promotes miR-302 Expression Leading to Self-renewal, Clonal Formation, and Cisplatin Resistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

Striking Anti-Multiple Myeloma (MM) Activity with the In Vitro Use of the Multikinase Inhibitor Sorafenib (S), Cytotoxic Synergy Between Bortezomib (B) and S, but Not Between B and Epigallocatechin-3-Gallate (EGCG; E) Enlarge the Present Knowledge In MM and Promise Novel Insights for Innovative Substance Exploitation In MM-Treatment

AbstractAbstract 5020 Introduction:As the development of novel anti-MM therapies is pursued worldwide in order to further improve survival in this disease, various innovative agents have to be eagerly tested in in vitro and in vivo models; the former being applied here. Bortezomib (B) has been shown to induce cell death in MMCLs and cytotoxic synergy with sorafenib (S) on various tumor cell lines. S, an oral multikinase inhibitor, targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. EGCG (E), one main green tea constituent, causes MM cell toxicity also, but seems to prevent tumor cell death induced by B in vitro and in vivo, this extend not being fully understood as yet. Methods:RPMI8226, U266 and L363 were cultured with RPMI1640/10% FCS. On day (d) 0, cells were treated with increasing concentrations of B, S and/or E. Cell viability and cytotoxicity were assessed on d3 and d6 via trypan blue and PI-staining. CD138 expression and morphologic changes were evaluated via FACS, immunocytochemistry and confocal microscopy. The effect of S on the chemotactic behaviour of L363 in response to conditioned media (CM = supernatant of M210B4 stromal cells) using 96-well chemotaxis chamber plates was also evaluated. Phosphorylation of ERK1/2 was determined by Western blot. The combined effect of S and B was determined using Calcusyn software: the resulting combination index (CI) defines additive effects (CI=1), synergism (CI<1) and antagonism (CI>1). Results:With 10 and 100μM S in L363, we observed increased median PI+ cells (62% and 94% on d3, respectively) as compared to the control (median PI+ d0: 11%), with similar increases on d6 (median 81% and 92%, respectively). In line with PI-observations, viable cells and CD138 expression substantially decreased in a dose- and time-dependent manner. After 3 days pre-incubation with increasing S-concentrations, MM cells were stained with Dapi, Phalloidin-Alexa-549 and CD138-FITC and analyzed by confocal microscopy: L363 cells highly expressed CD138 in the absence of S, whereas impressive CD138 downregulation, morphologic changes and reduction of F-actin content were observed with S-concentrations as low as 1μM. L363 cells exhibited a migratory response to CM, whereas after 3 days of preincubation with 10, 20 and 50μM S, L363 cells showed reduced migratory capacity in response to CM. Western blots showed a decrease in p-ERK1/2 expression levels after 24h inbubation of L363 cells with 10μM S. With 100nM B, PI in L363 increased from 11% on d0 to 84% on d3, albeit not as pronounced with 10nM B as was observed with 10μM S. E induced cytotoxicity in L363, particularly with 50 and 100μM, albeit - different to prior reports - B-induced cell death was preserved when the B-E-combination was tested: of note, however, after addition of increasing E-concentrations, no synergism or additive effect, rather than a plateau cytotoxic effect was observed. Combined B and S use showed synergism with 10nM and 10μM, respectively (CI=0.80). MMCLs stably co-expressing fluorescently labelled cytochrome C and histone H2 will allow the detection of induced apoptosis using live-cell imaging after anti-MM agent treatment. Conclusions:Our MM-based in vitro model revealed that B and S show remarkable therapeutic efficacy as single agents and synergism when combined, which confirms results in other tumor cell lines. E alone induced dose-dependent cell death and decreases in MM cell viability and when combined with B did neither synergize nor abolish B-induced cell death. Our results further enlarge the present knowledge in MM therapy and promise novel insights for innovative substances in the treatment of MM. Disclosures:No relevant conflicts of interest to declare.

Selective depletion of inducible HSP70 enhances immunogenicity of rat colon cancer cells.

Expression of inducible heat shock protein 70 (HSP70) in tumor cells has been proposed to enhance their immunogenicity. However, HSP70 has also been demonstrated to prevent tumor cell death, a key process for the development of tumor cell immunogenicity. In the present study, we investigated the influence of the HSP70 protein level on PRO colon cancer cell growth and immunogenicity in syngeneic BDIX rats and nude mice. These cells have a basal expression of HSP70 which can be substantially increased by heat shock. When injected subcutaneously in syngeneic animals, PRO cells do not induce any detectable immune response and give rise to progressive, metastatic and lethal tumors. Stable transfection of an anti-sense hsp70 cDNA in PRO cells (PRO-70AS cells) strongly decreased HSP70 expression and sensitized cell-free extracts to cytochrome c/dATP-mediated activation of caspases. Subcutaneous injection of PRO-70AS cells induced tumors that rapidly regressed in syngeneic rats while they grew normally in nude mice. Syngeneic rats injected with PRO-70AS cells became protected against a further challenge with PRO cells. The tumor-specific immune response induced by HSP70-depleted PRO-70AS cells was associated with an increased rate of cell death in vivo. These PRO-70AS cells were also more sensitive to NO-mediated, caspase-dependent, macrophage cytotoxicity in vivo. Altogether, these results indicate that reduced level of HSP70 expression in PRO- colon cancer cells results in the generation of a specific immune response by promoting cell death in vivo.

Expression of Bcl-2 in human breast cancer: correlation between hormone receptor status, p53 protein accumulation and DNA strand breaks associated with apoptosis.

The expression of Bcl-2, a suppressor of apoptotic cell death, was investigated in 52 invasive carcinomas of the breast using reverse transcription-polymerase chain reaction and immunohistochemical methods. After consideration of both sets of results, 42 tumors (80.8%) were confirmed to be positive (Bcl-2(+)) and 10 (19.2%) were judged negative (Bcl-2(-)) for Bcl-2 expression. Related factors (p53 protein accumulation, hormone receptor status and apoptotic cell index) were also examined using immunohistochemical and in situ end-labeling methods to elucidate their correlations with Bcl-2 expression. Bcl-2 expression correlated significantly with the hormone receptor status, whereas it showed significant inverse correlations with p53 accumulation and the apoptotic index. It was concluded that estrogen and mutant p53 are related to the regulation of Bcl-2 expression and that the ability to prevent tumor cell death due to Bcl-2 can be developed by breast cancers.