Decrease In Clonogenicity Research Articles

DIPG-04. TUMOR CELL-INTRINSIC TIM-3 PROMOTES DIFFUSE INTRINSIC PONTINE GLIOMA TUMORIGENESIS

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in the understanding of this disease, the median overall survival is still dismal. TIM-3 is emerging as an alternative to the classic immune checkpoints CTLA-4 and PD-1, and its expression regulates by various mechanisms the function of innate and adaptative immunity. In our work, we demonstrated that TIM-3 is expressed in murine and human DIPG tumor cell lines in vitro and in vivo. TIM-3 knockout using CRISPR/Cas9 was lethal in all human DIPG cell lines evaluated. However, a doxycycline-inducible shRNA-TIM-3 significantly reduced the proliferation of DIPG human cell lines (100% control vs. 25% shRNA TIM-3). Additionally, TIM-3KO murine cell lines displayed a significant decrease in clonogenicity, proliferation, and migration in vitro compared with parental DIPG murine cell lines. Interestingly, loss of TIM-3 expression did not result in cell death, as shown by the evaluation of apoptosis. In vivo evaluation of TIM-3KO cell lines tumorigenicity demonstrated that mice bearing TIM3-KO cells had an overall median survival (35 days) significantly higher than the ones bearing the control guide cells (16 days). RNAseq data indicated that TIM-3 plays a critical role in DIPG murine cells tumorigenesis and survival through the MAPK signaling pathway. Finally, single-cell sequencing and immunochemistry of human tumor samples uncovered TIM-3 expression in patients. TIM-3 expression in DIPG tumor cells correlated with genes positively involved in regulating ion transport, IL-6 production, and response to IFN-γ pathways. We also observed enrichment in genes regulating MAPK and ERK1/ERK2 cascades in these patients. Collectively, these data suggest that TIM-3 expression might have a role in the survival and tumorigenesis of DIPG cells.

Mesenchymal Stem Cell Therapy for Diabetic Kidney Disease: A Review of the Studies Using Syngeneic, Autologous, Allogeneic, and Xenogeneic Cells.

Diabetic kidney disease (DKD) is a microvascular complication of diabetes mellitus (DM) and comprises multifactorial pathophysiologic mechanisms. Despite current treatment, around 30-40% of individuals with type 1 and type 2 DM (DM1 and DM2) have progressive DKD, which is the most common cause of end-stage chronic kidney disease worldwide. Mesenchymal stem cell- (MSC-) based therapy has important biological and therapeutic implications for curtailing DKD progression. As a chronic disease, DM may impair MSC microenvironment, but there is compelling evidence that MSC derived from DM1 individuals maintain their cardinal properties, such as potency, secretion of trophic factors, and modulation of immune cells, so that both autologous and allogeneic MSCs are safe and effective. Conversely, MSCs derived from DM2 individuals are usually dysfunctional, exhibiting higher rates of senescence and apoptosis and a decrease in clonogenicity, proliferation, and angiogenesis potential. Therefore, more studies in humans are needed to reach a conclusion if autologous MSCs from DM2 individuals are effective for treatment of DM-related complications. Importantly, the bench to bedside pathway has been constructed in the last decade for assessing the therapeutic potential of MSCs in the DM setting. Laboratory research set the basis for establishing further translation research including preclinical development and proof of concept in model systems. Phase I clinical trials have evaluated the safety profile of MSC-based therapy in humans, and phase II clinical trials (proof of concept in trial participants) still need to answer important questions for treating DKD, yet metabolic control has already been documented. Therefore, randomized and controlled trials considering the source, optimal cell number, and route of delivery in DM patients are further required to advance MSC-based therapy. Future directions include strategies to reduce MSC heterogeneity, standardized protocols for isolation and expansion of those cells, and the development of well-designed large-scale trials to show significant efficacy during a long follow-up, mainly in individuals with DKD.

Abstract 1902: The comparative superiority of a bispecific antibody targeting alpha v and alpha 5 integrins is uniquely characterized by induced degradation of integrins

Abstract Background: Bispecific antibody blockade of alpha v and alpha 5 integrins (BsAbαvα5) resulted in superior anti-tumor activity in prostate cancer cells over monospecific αv (MsAbαv) and α5 blockade (MsAbα5), singly or in combination. We sought to define a molecular basis for this observation. Methods: We examined the comparative impact of monospecific and bispecific antibody strategies on membrane-specific and cellular expression of integrins as well as their transcription, translation and degradation fate in integrin av/a5+ prostate cancer cells (VCAP, C4-2b, DU-145 and PC-3). These dynamic changes were assessed in the context of functional studies including tumor-stromal interactions and downstream signaling. Results: With MsAbαv or MsAbα5 therapy, alone or in combination, upregulation of target integrin and/or the alternative integrin was observed which suggested both autoregulation as well as a cross-regulation among the target integrins. However, with BsAbαvα5 therapy, either a block in upregulation of target integrin/s or a marked decrease in cellular integrin expression was noted. Unexpectedly, these dynamic changes in cellular integrin expression with the BsAbαvα5 were retained up to 72 hours after antibody exposure with recovery of integrin expression in viable cells noted by 7 days. In cells recovered at 48-72 hours, a marked decrease in clonogenicity, adhesion and migration was observed. Flow cytometry indicated persistent membrane loss of integrin expression at 48-72 hours confirming that the impact of the BsAbαvα5 is not related to altered subcellular integrin trafficking alone. QRT-PCR studies did not identify upregulation of integrin transcription to account for upregulated integrin expression. Blockade of protein translation with cycloheximide failed to abrogate the differential impact of MsAbs or BsAbαvα5 on integrin expression. However, lysosomal inhibition with chloroquine blocked the downregulation of integrin expression with BsAbαvα5 therapy. Variable effects on downstream signaling in the Akt and MAPK pathway observed across cell types contrasted with consistent functional impairment of tumor-stromal interactions. Conclusions: BsAbαvα5 therapy uniquely induces lysosomal degradation of target integrins compared to MsAbs, alone or in combination. Cellular depletion of integrin expression serves as a pharmacodynamic marker of BsAbαvα5activity. By contrast, upregulated cellular expression secondary to MsAb therapy is likely secondary to a block in both integrin degradation and membrane recycling within the endosomal/lysosomal pathway. These observations provide additional insight into the mechanistic impact of the BsAbαvα5 as well as a pharmacodynamic method for monitoring in vivo activity. Citation Format: Raghav Joshi, Wenying Ren, Paul Mathew. The comparative superiority of a bispecific antibody targeting alpha v and alpha 5 integrins is uniquely characterized by induced degradation of integrins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1902.

Abstract 3299: Radiation induced p53-dependent cytoprotective autophagy fails to reduce radiation sensitivity in H460 non-small cell lung cancer cells

Abstract Our previous studies showed that radiation-induced autophagy could be either cytoprotective or nonprotective in A549 non-small cell lung cancer cells, HN30 and HN6 head and neck cancer cells and 4T1 breast tumor cells, and that this functional difference was related to the status of p53 in the cells (i.e. cytoprotective autophagy in p53 wild type (wt) cells and nonprotective autophagy in cells with mutant or null p53) [1]. Despite the fact that inhibition of autophagy has been proposed as a strategy to overcome resistance to radiation and chemotherapy, there is little direct information in support of the premise that the induction of autophagy confers resistance. As an example, studies in HCT-116 cells that are either wt or null in p53 demonstrate essentially identical radiation sensitivity [2], although the nature of radiation induced autophagy was not determined in these cell lines. To address this question, p53 wt and p53 null H460 cell lines were utilized to evaluate whether cytoprotective autophagy actually suppressed radiation sensitivity. Acridine orange staining, flow cytometric analysis and western blotting (LC3B, p62, Atg5) showed that following exposure to 6 Gy of radiation wtp53 and p53-null H460 cells exhibited a significant induction of autophagy. p53 wt H460 cells showed sensitization to radiation upon pharmacological autophagy inhibition and Atg5 knock down. However, pharmacological inhibition of autophagy and knock down of Atg5 genes failed to influence sensitivity to radiation in p53-null H460 cells. Furthermore, clonogenic survival assays showed that while exposure to radiation induced a dose-dependent (2, 4, 6, 8 Gy) decrease in clonogenicity in both p53wt and p53-null H460 cells, radiation sensitivity was not decreased in the p53 wt cells. Taken together, these findings indicate that cytoprotective autophagy may not actually alter the nature of radiation (and possibly chemotherapeutic) sensitivity in tumor cells. [1] Chakradeo, S, et al. Mol Pharm (2015): [2] Lindgren, T, et al. Int J Rad Biol (2015). Acknowledgments We would like to thank Dr. Santiago Lima (Virginia Commonwealth University) for the H460 p53-null cells. Citation Format: Jingwen Xu, Tareq Saleh, Liliya Tyutynuk-Massey, Yingliang Wu, David A. Gewirtz. Radiation induced p53-dependent cytoprotective autophagy fails to reduce radiation sensitivity in H460 non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3299. doi:10.1158/1538-7445.AM2017-3299

Pro-differentiating effects of a synthetic flavagline on human teratocarcinomal cancer stem-like cells.

As initiators of the carcinogenic process, cancer stem cells (CSCs) are considered as new targets for anti-cancer therapies. However, these cells are hidden in the cancer bulk and remain relatively insensitive to chemotherapy, which targets their proliferative capacities. Alternatively, growing evidences have pointed out that a differentiation therapy could adversely affect these cells, which consequently should lose their self-renewal properties and become less aggressive. In order to evaluate the differentiation potential of an emerging class of anti-cancer drugs, we used the poorly differentiated teratocarcinomal cell as a model of Oct4-expressing CSC and determined the molecular mechanisms induced by the highly active flavagline FL3. The drug, administrated at sublethal concentration and for long period, was able to downregulate the expression levels of the stemness factors Oct4 and Nanog at both transcriptional and translational levels, concomitantly with a decrease of clonogenicity. The appearance of specific neural markers further demonstrated the differentiation properties of FL3. Interestingly, an expression of active caspase-3 and an upregulation of the expression of the germ cell nuclear factor were observed in treated cells; this suggests that the suppression of Oct4 expression required for the induction of differentiation involves overlapping mechanisms of protein degradation and gene repression. Finally, this study shows that FL3, like all-trans retinoic acid (ATRA), acts as a differentiation inducer of teratocarcinomal cells. Thus, FL3 offers an alternative possibility for cancer treatment since it could target the carcinogenic process by inducing the differentiation of ATRA-resistant and Oct4-expressing CSCs, without toxic side effects on normal cells.

Sialidase NEU3 contributes neoplastic potential on colon cancer cells as a key modulator of gangliosides by regulating Wnt signaling.

The plasma membrane-associated sialidase NEU3 is a key enzyme for ganglioside degradation. We previously demonstrated remarkable up-regulation of NEU3 in various human cancers, with augmented malignant properties. Here, we provide evidence of a close link between NEU3 expression and Wnt/β-catenin signaling in colon cancer cells by analyzing tumorigenic potential and cancer stem-like characteristics. NEU3 silencing in HT-29 and HCT116 colon cancer cells resulted in significant decrease in clonogenicity on soft agar and in vivo tumor growth, along with down-regulation of stemness and Wnt-related genes. Analyses further revealed that NEU3 enhanced phosphorylation of the Wnt receptor LRP6 and consequently β-catenin activation by accelerating complex formation with LRP6 and recruitment of GSK3β and Axin, whereas its silencing exerted the opposite effects. NEU3 activity-null mutants failed to demonstrate the activation, indicating the requirement of ganglioside modulation by the sialidase for the effects. Under sphere-forming conditions, when stemness genes are up-regulated, endogenous NEU3 expression was found to be significantly increased, whereas NEU3 silencing suppressed sphere-formation and in vivo tumor incidence in NOD-SCID mice. Increased ability of clonogenicity on soft agar and sphere formation by Wnt stimulation was abrogated by NEU3 silencing. Furthermore, NEU3 was found to regulate phosphorylation of ERK and Akt via EGF receptor and Ras cascades, thought to be additionally required for tumor progression. The results indicate an essential contribution of NEU3 to tumorigenic potential through maintenance of stem-like characteristics of colon cancer cells by regulating Wnt signaling at the receptor level, in addition to tumor progression via Ras/MAPK signaling.

The CDX1–microRNA-215 axis regulates colorectal cancer stem cell differentiation

The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using high-throughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.

Rescue of fish exposed to a lethal dose of pathogen, by signals from sublethally exposed survivors.

Rainbow trout (Oncorhynchus mykiss, Walbaum) were challenged intraperitoneally with a sublethal dose of Vibrio anguillarum VIB1 and allowed to recover. Then, after 7 days, naïve fish, (designated as 'bystander' fish) which had never been exposed to the pathogen, were introduced to the same tank. These swam with the adapted (recovered) fish for 7 days before both groups and a control (never exposed directly to the pathogen or to recovered fish) group were exposed to a lethal dose of VIB1. Mortality records were 100% in the control group within 3 days, 47% in the adapted group and 60% in the unchallenged bystander group, which swam with the adapted group. In both the latter groups, the time to death of the non-surviving fish was attenuated. This inter-animal communication of signals has previously been documented for animals exposed to ionizing radiation. Assays of tissues from control, challenged and 'bystander fish exposed to the pathogen showed that a signal as yet unidentified but similar to that seen in bystanders to irradiated fish was being produced. This signal caused a sharp and transient increase in intracellular calcium and a decrease in clonogenicity in a well-characterized reporter assay.

Genetic and Pharmacologic Notch4 Inhibition Synergizes with FLT3 Tyrosine Kinase Inhibition in Vitro to More Effectively Eliminate FLT3/ITD-Positive Leukemia Cells

FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic stem/progenitor cells. Mutated in approximately 1/3 of patients, FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML). The presence of FLT3 internal tandem duplication (ITD) mutations, which renders FLT3 constitutively active, confers a particular poor prognosis. While several FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling, the clinical success of these drugs is limited because they fail to achieve frequent complete durable responses, despite achieving high levels of FLT3 inhibition. These limited clinical responses suggest that monotherapy is unlikely to be curative. Current clinical trials of FLT3 inhibitors are administered in combination with chemotherapy to achieve success in inducing complete remissions. While there is hope that FLT3 inhibition in combination with chemotherapy will increase cure rates, it would be optimal to achieve cures without chemotherapy altogether and its inherent toxicities. Towards this goal it is necessary to uncover critical genes/pathways that collaborate with FLT3 mutations to transform cells. In recent years, the use of whole genome sequencing has greatly expanded the list of genes mutated in AML. However, many of these mutations do not represent a practical therapeutic target because they are present in a low frequency in the patient population, making molecular targeted therapy for each of these improbable to achieve. We hypothesize that these mutated genes likely funnel into a more limited number of signaling pathways, with some pathways being more important than others in contributing to FLT3/ITD AML. Identifying FLT3/ITD cooperative pathways whose inhibition might synergize with FLT3 inhibition could hold promise for greatly improving the cure rate in FLT3 mutant AML.We performed a loss of function RNAi screen in FLT3/ITD+ AML cell lines (Molm14 and MV411) to uncover genes and pathways whose inhibition combined with FLT3/ITD inhibition to more effectively kill FLT3 mutant AML cells. Notch4 signaling was identified as one such potential target. We found Notch4 to be overexpressed in FLT3/ITD+ AML cells lines as well as in the Lin-Sca+Kit+ fraction of bone marrow isolated from FLT3/ITD+ mice. Stable, tetracycline-inducible Notch4 knockdown cell lines were established in Molm14 and MV411 cell lines. While Notch4 knockdown alone slightly decreased cell growth, Notch4 knockdown in combination with FLT3 TKI demonstrated a significant decrease in proliferation compared to FLT3 TKI treated cells expressing a scrambled shRNA control. Additionally, colony forming unit assays revealed that Notch4 knockdown cells treated with FLT3 TKIs exhibited a decrease in clonogenicity compared to FLT3 TKI treated control cells. Pharmacologic inhibition of Notch4 by treatment with gamma secreatse inhibition (GSI) was also tested. Molm14, MV411, and THP-1 (FLT3 wild type AML) cells were treated with FLT3 TKIs (sorafenib, CEP-701, or AC220) alone, GSI alone, FLT3 TKI plus GSI, or vehicle control and assessed for anti-proliferative and apoptotic effects by MTT, and annexin V/7-AAD staining, respectively. In both assays, the combination of FLT3 TKI and GSI exhibited synergy with combination index (CI) values <1. Furthermore, Western blot analysis showed that while treatment of Molm14 and MV411 cells with GSI had little effect on FLT3 signaling (pSTAT5, pAKT, pMAPK activation), the combination of GSI plus FLT3 TKI inhibited FLT3 signaling significantly more than treatment with FLT3 TKI alone. The results of these experiments identify Notch4 signaling as a potential pathway whose inhibition can synergize with FLT3 inhibition to more effectively kill FLT3/ITD+ AML cells. Combining FLT3 inhibition with Notch4 inhibition may have potential for improving the cure rate of patients with FLT3 mutant AML. DisclosuresNo relevant conflicts of interest to declare.

Donor's age dependent proliferation decrease of human bone marrow mesenchymal stem cells is linked to diminished clonogenicity.

While mesenchymal stem cells represent an interesting cell source for regenerative medicine, several points have to be investigated to improve their use in clinical, and in particular in the elderly population. This work studied the proliferation capacity of mesenchymal stem cells isolated from human bone marrow in function of donor's age. Doubling time after in vitro culture, clonogenicity and phenotype were analyzed in 17 samples ranging from 3 to 85 years old (mean 47 ± 27). Results showed an increase in the doubling time for cell coming from old donor compared to cells coming from young ones. This was accompanied by a decrease in clonogenicity while no changes were observe in cell phenotype. In conclusion, this study showed an effect of donor's age on the proliferation capacity of mesenchymal stem cells isolated from bone marrow that was correlated to a decrease in clonogenicity. The comprehension of molecular mechanism involved in this process could help to improve the clinical application of mesenchymal stem cells.

Quantitative Approaches to Detect Donor and Passage Differences in Adipogenic Potential and Clonogenicity in Human Bone Marrow-Derived Mesenchymal Stem Cells

Bone marrow-derived multipotent stromal cells (MSCs), also known as mesenchymal stem cells, have great promise due to their capacity for tri-lineage differentiation and immunosuppressive properties, which allows for their allogeneic use and ultimately may allow for treatment of many diseases. MSCs will require extensive expansion and passaging to obtain cells in sufficient numbers necessary for cell therapies. MSCs from many donors could potentially be used. Because of this, there is a need to understand the role of passaging and donor differences on differentiation capacity using quantitative approaches. Here, we evaluated MSCs from two donors (noted as PCBM1632 and PCBM1641 by the manufacturer) at tissue culture passages 3, 5, and 7. We used a colony forming unit (CFU) assay and limiting dilution to quantify clonogenicity and precursor frequency during adipogenesis, and quantitative real-time-polymerase chain reaction for adipogenic markers to evaluate changes on a gene expression level. Further, we observed changes in cell size, and we sorted small and large populations to evaluate size-related adipogenic potential. While the adipogenic precursor frequency of ∼1 in 76 cells remained similar through passages for cells from PCBM1641, we found a large decrease in the adipogenic potential of MSCs from PCBM1632, with 1 in 2035 cells being capable of differentiating into an adipocyte at passage 7. MSCs from both donors showed an increase in cell diameter with increasing passage, which correlates with a decrease in clonogenicity by CFU analysis. We also measured adipose lineage gene expression following induction of adipocyte differentiation. Expression of these genes decreased with passage number for MSCs from PCBM1632 and correlated with the decrease in adipogenic potential by passage 7. In contrast, MSCs from PCBM1641 showed increased expression of these genes with increasing passage. We have shown that several quantitative assays can detect differences in MSC differentiation capacity, clonogenicity, and cell size between donors and passages. These quantitative methods are useful to assess the quality of MSCs.

Overexpression of COX-2 in Celecoxib-Resistant Breast Cancer Cell Lines

Cyclooxygenase-2 (COX-2) plays a key role in breast cancer progression and metastasis. Effective therapeutic targeting of COX-2 would require the knowledge of whether a tumor is addicted to COX-2, and if we can counter the potential resistance to anti-COX-2 therapy. Herein we tested the hypothesis that celecoxib-resistance involves selection of cancer cells that overexpress COX-2. We selected celecoxib-resistant (CER) variants from two metastatic cell lines, SUM149 inflammatory breast cancer (IBC) cell line and MDA-MB-231-BSC60 cell line, by culturing them in the presence of celecoxib. We measured the relative levels of COX-2 protein and its network components Bcl-2, Bcl-xL, and Bax in the parental cell lines and their CER variants by Western blotting. To determine whether celecoxib resistance would increase tumorigenicity, we performed an in vitro clonogenicity assay. We determined the statistical significance of differences between the groups using the two-sample t-test. Both the celecoxib-resistant cell lines SUM149-CER and BSC60-CER produced significantly higher levels of COX-2 protein than their parental counterparts (P < 0.05). The CER variants produced a reduced level of pro-apoptosis protein Bax (both cell lines) and increased levels of anti-apoptosis proteins Bcl-2 (BSC60) or Bcl-xL (SUM149). Importantly, the CER variants had significantly higher clonogenicity than their parental cell lines (P < 0.05). The siRNA-mediated COX-2 knockdown in SUM149-CER cell line resulted in a significant decrease in clonogenicity and in Bcl-xL and Bcl-2 protein levels, thus supporting our hypothesis. Celecoxib-resistant variant cells present in breast cancer cell lines overexpress COX-2, which is robustly linked with survival pathways and clonogenicity. Since COX-2 is important in the variant cancer cells of aggressive nature, it represents a good therapeutic target.

A New Application for the Drug Tranilast: Effects on Breast Cancer Cell Proliferation, Migration, and Invasion

BackgroundTranilast (Rizaben®) has been used to treat allergic and auto‐immune diseases for the last 26 years. The objective of this study was to investigate whether tranilast also has an effect on breast cancer cell biological features relevant to cancer progression, based on the its mechanism of action which involves TGFβsignaling.MethodsCultures of the BT‐474 human breast cancer cell line were used. Cell proliferation, clonogenicity, cell migration, endoglin and MMP‐9 expression were assessed by: MTT assay, soft agar colony formation, wound assay and immunocytochemistry respectively.ResultsTranilast was found to inhibit breast cancer cell proliferation by 70%. In addition, the drug decreased by 54% the number and size of cell colonies grown in soft agar, consistent with a decrease in clonogenicity. The closure of wounds made in cancer cell monolayers was slowed (55%) by Tranilast, consistent with an inhibitory effect on tumor cell migration. Endoglin, which plays a role in TGFβ signaling, was downregulated by the drug. MMP‐9, which is a metalloproteinase involved in tumor invasion and metastasis was also decreased by tranilast.ConclusionsThese results indicate that tranilast exerts inhibitory effects on key biological features of human breast cancer cells. The results are consistent with an anti‐tumor effect which can be exploited in preclinical and clinical therapeutic trials.

Short-term exposure of multicellular tumor spheroids of a human glioma cell line to the glycolytic inhibitor 2-deoxy-D-glucose is more toxic than continuous exposure

The glycolytic inhibitor 2-deoxy-D-glucose (2-DG) has been used as a therapeutic agent and as an adjuvant in cancer therapy with either weekly fractions of the treatment or daily administration. While the weekly fraction has often been found to be nontoxic and effective, other treatment regimes are tolerated to a relatively lesser extent. It was therefore, considered worthwhile to investigate the efficacy of short- and long-term exposure of tumor cells to 2-DG under the controlled conditions. Seven-day-old MTS were exposed to 2-DG (5 mM, equimolar to glucose concentration in media) for different time intervals (30 min to 24 h) trypsinized and plated for clonogenicity. Alternatively, spheroids were grown either continuously in the presence of 2-DG or were treated with 2-DG for 2 h (short-term exposure) and grown in 2-DG-free media for 21 days and assessed for spheroid growth, cell viability, apoptosis, cytogenetic damage, mitochondrial status, and oxidative stress. Exposure of spheroids to 2-DG for 2-4 h induced 30% cell death (SF 0.70) while, a 24-h exposure resulted in only a marginal decrease in clonogenicity (SF 0.95). Furthermore, the spheroids disintegrated completely by 28 days in the case of 2-h exposure to 2-DG, while spheroids grown continuously in the presence of 2-DG repopulated. The cytotoxicity following short-term exposure of MTS to 2-DG was primarily due to the induction of apoptosis revealed by morphological features as well as flow cytometric analysis of the DNA content. Interestingly however, cytogenetic damage (micronuclei induction) was observed in spheroids that were continuously exposed to 2-DG. Short-term exposure to 2-DG resulted in a significant increase in ROS levels and a reduction in the levels of unoxidized cardiolipin as measured by NAO suggesting the involvement of mitochondria leakiness leading to oxidative stress which, could be responsible for apoptotic cell death observed under these conditions. However, continuous exposure to 2-DG resulted in a moderate level of oxidative stress leading to the genomic instability. Preliminary studies also show that spheroids exposed continuously to 2-DG result in the development of resistance to certain chemotherapeutic drugs which could be correlated with elevated levels of mdr1. The present results suggest that a persistent down-regulation of glycolysis (as seen here with continuous exposure to 2-DG) could activate prosurvival responses besides inducing moderate levels of oxidative stress resulting in the development of resistance against therapeutic agents.

Expression of the chemokine receptor CCR6 in the Lewis lung carcinoma (LLC) cell line reduces its metastatic potential in vivo

Chemokines and their receptors play important roles in various aspects of tumoral processes, and evidence was provided for their critical involvement in determining the metastatic destination of tumor cells. Here, we analyzed in vitro and in vivo, how CCR6 expression could alter the behavior of Lewis lung carcinoma (LLC) cells, which were shown to express low levels of the CCR6 ligand, CCL20 (LARC), both in vitro and in vivo. The expression of CCR6 significantly decreased the number of metastases in immunocompetent C57BL/6 mice, without affecting the tumor-forming ability of LLC cells. This was correlated with a decrease in clonogenicity in soft and hard agar, and with increased adhesion to type-IV collagen. These two observations made in basal conditions were enhanced when CCL20 was added to the assay medium. Thus, expression of CCR6 in tumor cells, associated with the local production of CCL20, decreased the metastatic potential of the LLC line. We propose a model, in which the expression of a chemokine receptor in tumor cells can act as a metastasis-suppressor, or a metastasis-promoting factor, according to the expression, or the absence of expression of the cognate ligand(s) in the tumor.

SEL1L expression in pancreatic adenocarcinoma parallels SMAD4 expression and delays tumor growth in vitro and in vivo.

Recent data suggest that SEL1L may play an important role in pancreatic carcinoma, similar to breast cancer, where the expression of SEL1L has been associated with a reduction in both proliferative activity in vitro and clinical tumor aggressiveness. To investigate this possibility, we examined the expression of Sel1L in a series of primary pancreatic carcinomas by immunohistochemistry and characterized the effects of Sel1L overexpression both in vitro and in vivo. In 74 pancreatic cancers analysed, 36% lacked Sel1L expression, although there was no significant correlation between the expression of Sel1L and any clinicopathologic parameter, including survival. However, immunohistochemical reactivity for Sel1L and Dpc4/Smad4 was concordant in 69% of cases (chi(2) test P&<0.004). Overexpression of SEL1L in stably transfected pancreatic cancer cells caused both a decrease in clonogenicity and anchorage-independent growth as well as a significant increase in the levels of activin A and SMAD4. When implanted in nude mice, Suit-2-SEL1L-overexpressing clones displayed a considerably reduced rate of tumor growth. Thus, it can be hypothesized that Sel1L plays an important function in the growth and aggressiveness of pancreatic carcinoma. Moreover, our data provide evidence that SEL1L has an impact on the expression of genes involved in regulation of cellular growth, possibly through the TGF-beta signaling pathway.

Inhibitory effect of p27KIP1 gene transfer on head and neck squamous cell carcinoma cell lines.

Although p27 gene mutations are rarely found in cancer, the level of p27 protein expression decreases during tumor development. In several tumors, including laryngeal cancer, decreased expression of p27 is associated with a poor prognosis. The proliferation-inhibitory effect of p27 gene transfer on human head and neck squamous cell carcinoma (HNSCC) cell lines (SNU-1041, SNU-1066, and SNU-1076) by adenoviral vector was investigated. On transduction of the human HNSCC cell line with adenovirus-p27 (Ad-p27), a high level of p27 expression and increase of cyclin D1 and E were observed. Cell cycle analysis showed a marked decrease in the proportion of cells in S phase and an increase in G1 phase. Soft agar clonogenic assay showed a marked decrease in clonogenicity. These results suggested that overexpression of p27 could show proliferation-inhibitory effects on HNSCC cell lines. Thus, gene therapy using adenovirus-p27 seemed to have a potential to develop into a new cancer gene therapy modality.

Involvement of caspases in 4-hydroxy-alkenal–induced apoptosis in human leukemic cells

4-Hydroxynonenal (HNE), a reactive and cytotoxic end-product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress–induced cell death and in various cell types has been shown to induce apoptosis. We have demonstrated that HNE, at micromolar concentrations, induces dose- and time-dependent apoptosis in a leukemic cell line (CEM-C7). Interestingly, much higher concentrations of HNE (>15-fold) were required to induce apoptosis in leukocytes obtained from normal individuals. We also demonstrate that HNE causes a decrease in clonogenicity of CEM-C7 cells. Furthermore, our data characterize the caspase cascade involved in HNE-induced apoptosis in CEM-C7 cells. Using specific fluorogenic substrates and irreversible peptide inhibitors, we demonstrate that caspase 2, caspase 3, and caspase 8 are involved in HNE-induced apoptosis, and that caspase 2 is the first initiator caspase that activates the executioner caspase 3, either directly or via activation of caspase 8. Our studies also suggest the involvement of another executioner caspase, which appears to be similar to caspase 8 but not caspases 2 and 3, in its specificity. The demonstration of decreased clonogenicity by HNE in the leukemic cells, and their higher susceptibility to HNE-induced apoptosis as compared to the normal cells, suggests that such compounds may have potential for leukemia chemotherapy.

Measurement of Sensitivity to Adriamycin in Hybrid Spheroids

Using the newly developed hybrid spheroid system, we have been able to measure sensitivities to adriamycin in two sublines of LoVo cells (a human colon adenocarcinoma line). Hybrid spheroids are composed of coagglomerated LoVo cells, and nonproliferating HeLa "feeder" cells, mimicking a population of minitumors. Spheroids of uniform size, but containing different numbers of LoVo cells, were incubated with various concentrations of adriamycin and after washing, plated for colony formation. Binomial counting statistics were achieved by marking surface-attached spheroids soon after plating, and scoring colonies one week later. From the fraction of noncolony formers, the average number of clonogens per spheroid (clonogenicity) was calculated using the zero term of the Poisson distribution. The effect of treatment with adriamycin was evaluated from the decrease in clonogenicity in parallel series of hybrid spheroids. Response to treatment was found to be independent of cellular clonogenicity in the control, untreated series. Dose-response curves were obtained for each of the two sublines after treatment for 1.5 h with various concentrations of adriamycin. The difference in the response to adriamycin between the two sublines amounted to a factor of 27 (deduced from D0 ratios; these are the same as isosurvival dose ratios).

Cloning efficiency of human melanoma cells is modulated after invasion through a reconstituted basement membrane

Three human malignant melanoma cell strains (C8146A, C8146C, and C83-2CY), three established human melanoma cell lines (A375P, A375M, and C8161), and one selected human melanoma subline (A375P-5) were studied to determine if invasion through a reconstituted basement membrane-coated filter (RBMF), which selects the most aggressively invasive cells, would also modulate the cloning efficiency of these cells in soft agar. With the use of the Membrane Invasion Culture System (MICS), all cell strains tested showed a significant increase in cloning efficiency (1.05–9.3-fold) following transit through the RBMF when compared to unmanipulated populations. The established cell lines (A375P, A375M, and C8161) and the A375P-5 subline showed either a decrease or unaltered status in cloning efficiency after invasion. However, all cells demonstrated a consistent decrease in clonogenicity following transit through an uncoated filter compared with RBMFs, thus suggesting the influence of the extracellular matrix on tumor cell clonogenic properties. In general, the established cell lines were more clonogenic before invasion of the RBMF compared with the cell strains, and no correlation was found between clonogenic potential and invasive or metastatic capability. These data may provide important insight into the underlying mechanisms of tumor cell invasion and the subsequent formation and dissemination of metastases in vivo.