Inhibited Cell Motility Research Articles

FoxO3a Inhibits Tamoxifen-Resistant Breast Cancer Progression by Inducing Integrin α5 Expression.

Simple SummaryTamoxifen is a mainstay of adjuvant treatment for estrogen receptor α-positive (ERα+) breast tumors, which account for over 70% of all the diagnosed breast malignancies. Unfortunately, ~30% of patients initially responding to the therapy will eventually relapse with an endocrine-resistant disease. Here, we show that the transcription factor FoxO3a can overcome tamoxifen resistance by inhibiting cell motility and invasiveness, the main features of tumor progression. The underlying mechanism could be ascribed to FoxO3a-dependent transcriptional up-regulation of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion. Indeed, FoxO3a and integrin α5 expression is significantly correlated in ERα+ tumors, and FoxO3a protective effects are lost in cells with reduced levels of integrin α5. Therefore, a pharmacological increase in FoxO3a levels could represent an effective approach to be exploited in combination with tamoxifen treatment in order to restore the sensitivity to the therapy in refractory tumors.Resistance to endocrine therapy is still a major clinical challenge in the management of estrogen receptor α-positive (ERα+) breast cancer (BC). Here, the role of the Forkhead box class O (FoxO)3a transcription factor in tumor progression has been evaluated in tamoxifen-resistant BC cells (TamR), expressing lower levels of FoxO3a compared to sensitive ones. FoxO3a re-expression reduces TamR motility (wound-healing and transmigration assays) and invasiveness (matrigel transwell invasion assays) through the mRNA (qRT-PCR) and protein (Western blot) induction of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion and signaling. The induction occurs through FoxO3a binding to a specific Forkhead responsive core sequence located on the integrin α5 promoter (cloning, luciferase, and ChIP assays). Moreover, FoxO3a failed to inhibit migration and invasion in integrin α5 silenced (siRNA) cells, demonstrating integrin α5 involvement in both processes. Finally, using large-scale gene expression data sets, a strong positive correlation between FoxO3a and integrin α5 in ERα+, but not in ER-negative (ERα−), BC patients emerged. Altogether, our data show how the oncosuppressor FoxO3a, by increasing the expression of its novel transcriptional target integrin α5, reverts the phenotype of endocrine-resistant BC toward a lower aggressiveness.

Amyloid β aggregation induces human brain microvascular endothelial cell death with abnormal actin organization

Cerebral amyloid angiopathy (CAA) is a disease in which amyloid β (Aβ) is deposited on the walls of blood vessels in the brain, making those walls brittle and causing cerebral hemorrhage. However, the mechanism underlying its onset is not well understood. The aggregation and accumulation of Aβ cause the occlusion and fragility of blood vessels due to endothelial cell damage, breakdown of the blood-brain barrier, and replacement with elements constituting the blood vessel wall. In this study, we observed the effect of Aβ on human primary brain microvascular endothelial cells (hBMECs) in real-time using quantum dot nanoprobes to elucidate the mechanism of vascular weakening by Aβ. It was observed that Aβ began to aggregate around hBMECs after the start of incubation and that the cells were covered with aggregates. Aβ aggregates firmly anchored the cells on the plate surface, and eventually suppressed cell motility and caused cell death. Furthermore, Aβ aggregation induced the organization of abnormal actin, resulting in a significant increase in intracellular actin dots over 10 μm2. These results suggest that the mechanism by which Aβ forms a fragile vessel wall is as follows: Aβ aggregation around vascular endothelial cells anchors them to the substrate, induces abnormal actin organization, and leads to cell death.

Serum Proteomic Analyses Suggest That the HMGB1 and Other Inflammatory Pathways Are Operational in MBL and Are Less in Overt CLL

Background. CLL-like monoclonal B-ceII lymphocytosis (MBL) is considered a requisite precursor of CLL, with 1-2% of subjects annually progressing to CLL requiring therapy. The role of immune alterations leading to and operating in MBL and controlling progression to CLL is not well characterized. Since an increased frequency of immune-related conditions associated with immune dysfunction exists prior to CLL diagnosis (Landgren et al, Br J Haematol 2007 and Blood 2007; Andersen et al, Leukemia 2020), immune alterations likely exist in MBL. We have examined the serum protein profiles of MBL and CLL in search of clues linking immune dysfunction with malignant transformation.Objectives. 1. Characterize the serum proteomic profiles of MBL individuals, IGHV-mutated CLL (M-CLL) and IGHV-unmutated CLL (U-CLL) patients, and age-matched healthy controls (HC). 2. Compare the serum proteomic profiles between the groups. 3. Assess the effect of IGHV mutation status on serum proteomic profiles in CLL.Methods. A total of 12 MBL, 12 M-CLL, 12 U-CLL and 12 age-matched HC were studied. Patients were cared for at Northwell Health, New York (n=25) and Hospital del Mar, Barcelona (n=11). All patients were treatment naive except for one U-CLL patient who was treated one year beforehand. Serum samples were collected, and their protein levels measured employing SOMAmers (modified single-stranded DNA aptamers; SomaLogic) to quantify relative levels of 1,310 proteins. P-values <0.05 were used to define significantly different protein levels between groups. Ingenuity Pathway Analysis (IPA; QIAGEN) was employed to evaluate potential protein interactions and pathways. IPA pathways with P-value <0.05 and Z-score ≥2 or ≤-2 were considered to be activated or inhibited, respectively.Results. Overall, the levels of 862 proteins differed significantly between groups: MBL vs. HC: 10 downregulated (d) and 24 upregulated (u); M-CLL vs. HC: 206d and 6u; U-CLL vs. HC: 54d and 40u; M-CLL vs. MBL: 384d and 5u; U-CLL vs. MBL: 74d and 24u; and M-CLL vs. U-CLL: 35d and 0u. IPA highlighted a role for the pro-inflammatory HMGB1 pathway in several comparisons. First, an activated HMGB1 pathway was predicted in MBL compared to HC, together with activation of phagocytes, and an inhibition of the systemic immune suppressor TGF-β. Second and consistent with the former, U-CLL patients displayed an activated HMGB1 pathway compared to HC, along with other signs of immune stimulation (activated NFkB and Th1 pathways, maturation of dendritic cells, and inhibition of TGF-β) and leukemic progression (activated progression of tumor, and leukocyte extravasation). Third and contrary to the above, the HMGB1 pathway was inhibited when comparing M-CLL to HC, in line with a global immune suppression signature (inhibited PRR, GM-CSF, FcεRI, IL1, IL8, TNF, Th1, STAT3 and NFkB pathways, in addition to inhibited cell movement, viability and activation). Notably, inhibition of immune pathways was predicted for both M-CLL and U-CLL compared to MBL (diminished TNF and IL6 signaling, and reduced cell movement), although the greatest differences were seen for M-CLL vs. MBL comparison, including blockade of the HMGB1 pathway in M-CLL. Finally, the M-CLL vs. U-CLL comparison suggested inhibited INFγ, IL2, IL3, IL4, NFkB, and decreased T lymphocyte stimulation and movement of tumor cells in M-CLL patients.Conclusions. An increased pro-inflammatory signature with involvement of the HMGB1 pathway was identified in MBL and U-CLL compared with HC, whereas the opposite was seen for M-CLL. Since MBL most often exhibit mutated IGHV (91% of cases in our cohort), these findings suggest immune stimulation as a characteristic feature in the pre-leukemic and U-CLL leukemic stage that surprisingly is not operative in M-CLL. Consistent with this, M-CLL displayed a global immune suppression (HMGB1 pathway inhibition), whereas U-CLL exhibited signs of immune stimulation (HMGB1 pathway activation) compared to HC which may relate to distinct capabilities of the two subtypes to interact with the microenvironment. Lastly, the increased inflammatory signature identified in MBL, which are mainly IGHV-mutated, was lessened in CLL, mainly in M-CLL patients. This is consistent with a decreased influence of immune imbalance and the HMGB1 pathway associated with IGHV-mutated clonal expansions.Acknowledgments. GB is supported by a grant from Fundación Alfonso Martín Escudero. DisclosuresAllen: Alexon: Research Funding; Bristol Myers Squibb: Other: Equity Ownership; C4 Therapeutics: Other: Equity Ownership; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees. Rhodes: Conquer Cancer Foundation Young Investigator Award: Other: Grant/Research Support; AbbVie, Genentech, Pharmacyclics, TG Therapeutics: Other: Consultant.

Cell-to-cell heterogeneity in Sox2 and Bra expression guides progenitor motility and destiny.

Although cell-to-cell heterogeneity in gene and protein expression within cell populations has been widely documented, we know little about its biological functions. By studying progenitors of the posterior region of bird embryos, we found that expression levels of transcription factors Sox2 and Bra, respectively involved in neural tube (NT) and mesoderm specification, display a high degree of cell-to-cell heterogeneity. By combining forced expression and downregulation approaches with time-lapse imaging, we demonstrate that Sox2-to-Bra ratio guides progenitor's motility and their ability to stay in or exit the progenitor zone to integrate neural or mesodermal tissues. Indeed, high Bra levels confer high motility that pushes cells to join the paraxial mesoderm, while high levels of Sox2 tend to inhibit cell movement forcing cells to integrate the NT. Mathematical modeling captures the importance of cell motility regulation in this process and further suggests that randomness in Sox2/Bra cell-to-cell distribution favors cell rearrangements and tissue shape conservation.

Cytoplasmic pressure maintains epithelial integrity and inhibits cell motility

Cytoplasmic pressure, a function of actomyosin contractility and water flow, can regulate cellular morphology and dynamics. In mesenchymal cells, cytoplasmic pressure powers cell protrusion through physiological three-dimensional extracellular matrices. However, the role of intracellular pressure in epithelial cells is relatively unclear. Here we find that high cytoplasmic pressure is necessary to maintain barrier function, one of the hallmarks of epithelial homeostasis. Further, our data show that decreased cytoplasmic pressure facilitates lamellipodia formation during the epithelial to mesenchymal transition (EMT). Critically, activation of the actin nucleating protein Arp2/3 is required for the reduction in cytoplasmic pressure and lamellipodia formation in response to treatment with hepatocyte growth factor (HGF) to induce EMT. Thus, elevated cytoplasmic pressure functions to maintain epithelial tissue integrity, while reduced cytoplasmic pressure triggers lamellipodia formation and motility during HGF-dependent EMT.

Melittin suppresses epithelial-mesenchymal transition and metastasis in human gastric cancer AGS cells via regulating Wnt/BMP associated pathway.

Gastric cancer has a poor prognosis; once cancer has metastasized, it can easily lead to patient death. Melittin is one of the major components extracted from the bee venom. It has been shown that melittin emerges antitumor activities against many human cancer cell lines. Our results indicated that melittin at 0.2-0.5 µm significantly reduced total cell viability in human gastric cancer AGS cells. At low concentrations (0.05-0.15 µm), melittin displayed antimetastasis effects and inhibited cell adhesion and colony formation. Besides, it inhibited cell motility and suppressed cell migration and invasion. Melittin inhibited the activities of MMP-2 and MMP-9 and the integrity of cell membrane in AGS cells. Furthermore, Western blotting results showed that melittin decreased the protein expressions of Wnt/BMP and MMP-2 signaling pathways. Based on these observations, melittin inhibited cell migration and invasion of AGS cells through multiple signaling pathways. It may be used to treat metastasized gastric cancers in the future.

Campylobacter jejuni Triggers Signaling through Host Cell Focal Adhesions To Inhibit Cell Motility.

ABSTRACTCampylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblotting, and superresolution interferometric photoactivated localization microscopy (iPALM). We found that C. jejuni infection of epithelial cells results in increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction in collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of a protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially reestablishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions.

PolyI:C suppresses TGF-β1-induced Akt phosphorylation and reduces the motility of A549 lung carcinoma cells.

Epithelial mesenchymal transition (EMT) is a critical process involved in the invasion and metastasis of cancer, including lung cancer (LC). Transforming growth factor (TGF)-β is one of factors capable of inducing EMT. Polyinosinic-polycytidylic acid (polyI:C), a synthetic agonist for toll-like receptor (TLR) 3, can enhance immune responses and has been used as an adjuvant for cancer vaccines; however, it remains unclear whether it influences other process, such as EMT. In the present study, we examined the effects of polyI:C on TGF-β-treated A549 human LC cells. By in vitro cell proliferation assay, polyI:C showed no effect on the growth of A549 cells treated with TGF-β1 at the concentration range up to 10μg/ml; however, it markedly suppressed the motility in a cell scratch and a cell invasion assay. By Western blotting, polyI:C dramatically decreased TGF-β1-induced Ak strain transforming (Akt) phosphorylation and increased phosphatase and tensin homologue (PTEN) expression without affecting the Son of mothers against decapentaplegic (Smad) 3 phosphorylation or the expression level of E-cadherin, N-cadherin or Snail, indicating that polyI:C suppressed cell motility independently of the 'cadherin switching'. The Akt inhibitor perifosine inhibited TGF-β1-induced cell invasion, and the PTEN-specific inhibitor VO-OHpic appeared to reverse the inhibitory effect of polyI:C. PolyI:C has a novel function to suppress the motility of LC cells undergoing EMT by targeting the phosphatidylinositol 3-kinase/Akt pathway partly via PTEN and may prevent or reduce the metastasis of LC cells.

EMID1, a multifunctional molecule identified in a murine model for the invasion independent metastasis pathway

EMI Domain Containing 1 (EMID1) was identified as a potential candidate metastasis-promoting gene. We sought to clarify the molecular function of EMID1 and the protein expression. Overexpression and knockdown studies using mouse tumor cell lines identified two novel functions of EMID1: intracellular signaling involving enhancement of cell growth via cell cycle promotion and suppression of cell motility, and inhibition of cell–matrix adhesion by extracellularly secreted EMID1. EMID1 deposited on the culture dish induced self-detachment of cells that overexpressed the protein and inhibited adhesion of additionally seeded cells. This multifunctional property involving both intracellular signaling and the extracellular matrix suggests that EMID1 may be a matricellular proteins. Expression analysis using immunohistochemical staining revealed expression of EMID1 that was limited to chief cells of the gastric fundic gland and β cells of the pancreatic islets in normal adult human tissues, implying cell-specific functions of this molecule. In addition, increased expression of EMID1 protein detected in some cases of human cancers implies that EMID1 might be a new therapeutic target for cancer treatment.

Timing of Novel Drug 1A-116 to Circadian Rhythms Improves Therapeutic Effects against Glioblastoma.

The Ras homologous family of small guanosine triphosphate-binding enzymes (GTPases) is critical for cell migration and proliferation. The novel drug 1A-116 blocks the interaction site of the Ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase with some of its guanine exchange factors (GEFs), such as T-cell lymphoma invasion and metastasis 1 (TIAM1), inhibiting cell motility and proliferation. Knowledge of circadian regulation of targets can improve chemotherapy in glioblastoma. Thus, circadian regulation in the efficacy of 1A-116 was studied in LN229 human glioblastoma cells and tumor-bearing nude mice. Methods. Wild-type LN229 and BMAL1-deficient (i.e., lacking a functional circadian clock) LN229E1 cells were assessed for rhythms in TIAM1, BMAL1, and period circadian protein homolog 1 (PER1), as well as Tiam1, Bmal1, and Rac1 mRNA levels. The effects of 1A-116 on proliferation, apoptosis, and migration were then assessed upon applying the drug at different circadian times. Finally, 1A-116 was administered to tumor-bearing mice at two different circadian times. Results. In LN229 cells, circadian oscillations were found for BMAL1, PER1, and TIAM1 (mRNA and protein), and for the effects of 1A-116 on proliferation, apoptosis, and migration, which were abolished in LN229E1 cells. Increased survival time was observed in tumor-bearing mice when treated with 1A-116 at the end of the light period (zeitgeber time 12, ZT12) compared either to animals treated at the beginning (ZT3) or with vehicle. Conclusions. These results unveil the circadian modulation in the efficacy of 1A-116, likely through RAC1 pathway rhythmicity, suggesting that a chronopharmacological approach is a feasible strategy to improve glioblastoma treatment.

Abstract 267: Light-activated molecular nanomachines kill bladder cancer cells

Abstract Introduction: Molecular nanomachines (MNMs) have been studied for their anti-tumor efficacy in a number of solid malignancies. They consist of small (1 nm) organic molecule-based motors that change conformation upon light activation and mechanically drill holes in the cell membrane resulting in cellular necrosis, representing a novel form of molecular mechanical therapy. Recently, we generated a blue light spectrum (395-405 nm)-activated MNMs which showed promising results in pancreatic cancer cell lines. Herein, we evaluate the in vitro efficacy in urothelial carcinoma, a disease characterized by high rates of recurrence, progression, and therapeutic resistance. Methods: MNMs activated by the blue light spectrum were synthesized. In vitro efficacy of the activated MNMs (8 µM in 0.1% DMSO) was investigated in TCCSUP and UC-3 human bladder cancer cell lines. Cells were incubated with MNMs for 30 minutes, then treatment groups were subjected to 300 mW/cm2 blue light (comparable to Blue Light Cystoscope) for 4 minutes. Control groups included MNMs without light, 0.1% DMSO (required for solubility of the MNMs) with light, and 0.1% DMSO without light. Cell viability was quantitatively evaluated utilizing propidium iodide and flow cytometry assay with each datapoint representing triplicates. Additionally, cells were grown and subjectively tested for cell adhesion to the culture dish at 1d following light and MNM treatment. In addition, we utilized the wound healing assay to detect changes in cell motility following MNM treatment. Results: TCCSUP cells exhibited almost 100% loss in viability when treated with light-activated MNMs. Solution only (0.1% DMSO) and unactivated MNMs exhibited less than 5% change in cell viability, whereas, solution and light exhibited 20% reduction in cell viability suggesting some light sensitivity of TCCSUP cells. Cell adhesion assay 1d post-treatment confirmed 100% cell death for the MNMs + light treated cells, while showing survival of cells with light only treatment. In addition, results from wound healing assay conducted on UC-3 cells demonstrated at 72 h, there was significant inhibition of cell motility in the treatment group (MNMs + light) compared to control groups. Conclusions: Light-activated MNM exhibit in vitro efficacy in multiple human bladder cancer cells lines. MNMs are a promising novel therapeutic modality for the intravesical treatment of bladder cancer, and there are no known resistance mechanisms. Further preclinical characterization of antitumor efficacy and tolerability are necessary in in vivo models. Citation Format: Supriya Nagaraju, Alexis van Venrooy, Patrick J. Hensley, Kelly K. Bree, Ciceron Ayala-Orozco, Nathan A. Brooks, David Izhaky, James M. Tour, Ashish M. Kamat. Light-activated molecular nanomachines kill bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 267.

Abstract 46: A new class of precision therapeutics that inhibit prostate cancer mediated bone destruction

Abstract The destructive nature of bone metastases dominates the clinical management of advanced prostate cancer (PCa) and is a common cause of morbidity and mortality. Our current ability to therapeutically target this process is limited. Here we describe the synthesis and functional characterization of the first-in-class Dual Acting Bone Defending agents (DABDs), which couple a small molecule therapeutic that inhibits cell movement to bone trophic bisphosphonates. Upfront in-vitro screens assessed DABD efficacy at inhibiting PCa cell migration and invasion and osteoclast mediated bone destruction. Systemic models of PCa metastasis, inclusive of intracardiac (IC) and intratibial (IT) injection models, were deployed to characterize tolerance and efficacy in-vivo. Finally, in-vitro characterization of DABD molecular mechanism of action was performed, as guided by the existing knowledge base related to the parental compounds. Results demonstrated that DABDs inhibit PCa cell migration and invasion, chemically bind hydroxyappetite, and inhibit osteoclast mediated bone destruction in vitro. Following IC injection of PCa cells, a model where cells are placed into circulation, metastasize to bone, grow and induce bone destruction, emulating the clinical scenario in humans, DABD treatment inhibited circulating PCa cells from forming new bone metastasis and prolonged life in a dose-dependent fashion. In the IT mouse models of established metastatic lesions, DABDs induce reversion of PCa cell growth and disrupt further bone destruction. DABDs at doses 1000 fold higher than those associated with efficacy are well tolerated. At the molecular level, DABDs retain the molecular mechanisms of action of their constituent chemicals. They inhibit farnesyl diphosphate synthase (FDPS), in the mevalonate pathway, leading to decreased prenylation of small GTPases, including the Ras related protein, Rap-1A, as well as inhibit Raf1 activation, inducing apoptosis and impaired cell movement, respectively. DABDs are a new class of agents that inhibit formation of new bone metastasis, revert growth of established metastasis, prolong life, and have a high potential for improving clinical outcome in humans. Citation Format: Ryan R. Gordon, Abhinandan Pattanayak, Wenqi Li, Raymond Bergan. A new class of precision therapeutics that inhibit prostate cancer mediated bone destruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 46.

Retracted: Silencing of cystatin SN abrogates cancer progression and stem cell properties in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) accounts for approximately 80% of total thyroid cancers worldwide. Although the prognosis for early‐stage PTC is favorable, the 5‐year survival rate of patients with late‐stage PTC is still very poor. Cystatin SN (cystatin 1, CST1) facilitates the progression of multiple cancers, but its role in regulating PTC pathogenesis is still largely unknown. In this study, we measured the expression levels of CST1 in PTC clinical tissues and cell lines by real‐time quantitative PCR and western blot analysis, and we performed gain‐ and loss‐of‐function experiments to examine the effects of CST1 on PTC cell growth, invasion, migration, epithelial–mesenchymal transition and stemness. Tumorigenicity was assessed using in vivo tumor‐bearing nude mouse models. As expected, upregulated CST1 was observed in PTC tissues (P < 0.05) and cells, compared with their normal counterparts (P < 0.05); furthermore, patients with PTC with higher levels of CST1 exhibited unfavorable prognosis (P < 0.05). In addition, CST1 ablation inhibited PTC cell growth (P < 0.05) in vivo and in vitro. Silencing of CST1 also inhibited cell motility and epithelial–mesenchymal transition in PTC cells (P < 0.05), whereas CST1 overexpression had the opposite effects on the earlier cellular functions. Notably, up‐regulation of CST1 promoted cell spheroid formation (P < 0.05) and increased the expression levels of stemness signatures (P < 0.05) in PTC cells. Collectively, these findings suggest that CST1 functions as an oncogene to facilitate cancer development and promote cancer stem cell properties in PTC cells, increasing our understanding of PTC pathogenesis mechanisms and possibly aiding in the development of potential therapeutic strategies.

HMT Exerts an Anticancer Effect by Targeting PAK-1

Heamatang (HMT) is a classic medicinal formula used in traditional Chinese and Korean medicine; it contains seven distinct components, mainly of herbal origin. HMT is used as an antiaging remedy, treating urinary disorders and increasing energy and vitality. However, the therapeutic applications of this formula have not been evaluated with evidence-based science. Therefore, we assessed HMT through various in vitro methods, including cell viability assay, fluorescence-activated cell sorting assay (FACS), Western blotting, migration assay, three-dimensional (3D) cell culture, siRNA-mediated PAK-1 knockdown, and crystal violet assays. HMT decreased PAK-1 expression in PC-3 cells and inhibited cell viability, growth, and motility. The inhibition of cell motility by HMT was correlated with PAK-1-mediated inhibition of Lim domain kinase (LIMK) and cofilin. HMT induced G1 arrest and apoptosis through the transcriptional regulation of cell cycle regulatory proteins and apoptosis-related proteins (increase in c-cas3 and inhibition of PARP and BCL-2). Moreover, HMT suppressed PAK-1 expression, leading to the inhibition of AKT activities. Finally, we showed that decursin was the active ingredient involved in the inhibitory effect of HMT on PAK-1. Our findings demonstrated that HMT exerts its anticancer influence through the inhibition of PAK-1. The HMT formula could be applied in various fields, including functional health food and pharmaceutical development.

Anti-Tumoral Effects of a (1H-Pyrrol-1-yl)Methyl-1H-Benzoimidazole Carbamate Ester Derivative on Head and Neck Squamous Carcinoma Cell Lines.

Nocodazole is an antineoplastic agent that exerts its effects by depolymerizing microtubules. Herein we report a structural analog of nocodazole, a (1H-pyrrol-1-yl)methyl-1H-benzoimidazole carbamate ester derivative, named RDS 60. We evaluated the antineoplastic properties of RDS 60 in two human head and neck squamous cell carcinoma (HNSCC) cell lines and we found that this compound significantly inhibited replication of both HNSCC cell lines without inducing any important cytotoxic effect on human dermal fibroblasts and human keratinocytes. The treatment of HNSCC cell lines with 1 μM RDS 60 for 24 h stopped development of normal bipolar mitotic spindles and, at the same time, blocked the cell cycle in G2/M phase together with cytoplasmic accumulation of cyclin B1. Consequently, treatment with 2 μM RDS 60 for 24 h induced the activation of apoptosis in both HNSCC cell lines. Additionally, RDS 60 was able to reverse the epithelial-mesenchymal transition and to inhibit cell migration and extracellular matrix infiltration of both HNSCC cell lines. The reported results demonstrate that this compound has a potent effect in blocking cell cycle, inducing apoptosis and inhibiting cell motility and stromal invasion of HNSCC cell lines. Therefore, the ability of RDS 60 to attenuate the malignancy of tumor cells suggests its potential role as an interesting and powerful tool for new approaches in treating HNSCC.

CRGDyK-modified procaine liposome inhibits the proliferation and motility of glioma cells via the ERK/p38MAPK pathway.

Glioma is a common type of primary tumor in the central nervous system. Glioma has been increasing in incidence yearly and is a serious threat to human life and health. The aim of the present study was to prepare liposomes for enhanced penetration of the blood-brain barrier and targeting of glioma. A procaine-loaded liposome modified with the cyclic pentapeptide cRGDyK (Pro/cRGDyK-L) was designed and developed. The particle size, ζ potential, encapsulation efficiency, release profile, stability and hemolysis of Pro/cRGDyK-L were characterized in vitro. The targeting and antitumor effects of Pro/cRGDyK-L were also investigated in vitro and in vivo. The results suggested that the cRGDyK peptide significantly facilitated the ability of liposomes to transfer procaine across the BBB and improved the cellular uptake of procaine by C6 glioma cells. The results further demonstrated that Pro/cRGDyK-L strongly suppressed cell motility, stimulated apoptosis and induced cell cycle arrest. The findings further confirmed that Pro/cRGDyK-L exhibited superior antitumor effects by targeting the ERK/p38MAPK pathway and thereby suppressed tumor growth in mice. In conclusion, the present study indicated the potential of Pro/cRGDyK-L as a means to provide improved therapeutic effects on glioma through the ERK/p38MAPK pathway.

New tropolone derivative to inhibit cell motility in colorectal cancer cells.

e15043 Background: Tropolones are small, naturally occurring organic compounds that have anti-cancer effects in a variety of cancer cell lines. Among proposed mechanisms of tropolone derivatives anti-cancer activity are caspase activation, matrix metalloproteinases and histone deacetylases inhibition and other actions. Since matrix metalloproteinases are the major proteins that contribute to tumor cell tissue invasion and migration their blockers are extensively studied as prospective anti metastatic agents. We assumed that tropolones may also have potential as metastases prevention drugs. The aim of the study was to investigate the influence of new tropolone derivative 2-(6,8-dimethyl-5-nitro-4-chloroquinolin-2-yl)-5,6,7-trichloro-1,3-tropolone on the cell migration capacity in colorectal cancer cells. Methods: Cell migration in permanent colorectal cancer cell lines HT29, Caco2 and HCT116 was studied using the standard wound healing assay with automated image acquisition and analysis on Lionheart FX (BioTek) cell imager. Cell cultures were maintained in DMEM medium with L-glutamine (1 μM) (Gibco) and 10% FBS (Gibco) at 37C0 and 5.0% CO2. The day before assay establishment cells were seeded at 250 000 cells per well on 24-well plates. After attachment of cells, a vertical wound in cell layer was made manually in each well, and then medium with studied tropolone derivative (5 μM) was added. Plates with cells were continuously incubated and photographed in cell imager at 37C0 and 5.0% CO2. The extent of cells migration was measured as the percent of wound area decrease after 48 hours of incubation in relation to starting time point. Data are given as: Mean ± 95% confidence interval. Results: The new tropolone derivative under study exhibited pronounced anti migratory activity in all studied colorectal cancer cell cultures. The most prominent effect was observed in HCT116 cell line: after 48 hours of incubation relative wound area decrease was 48.2%±5.7% in control and 7.2%±3.15% in experimental groups. In Caco2 cell line cell migration inhibition was the least with 65%± 6.9% relative wound area decrease in control and 51.5%± 7.7% in experimental groups. The moderate effect was observed in HT29 cell line (measured parameter was 63.6%±7.6% and 36.9%±11.6% in control and experimental groups accordingly). The obtained difference between control and experimental groups was significant in all tested cell cultures at 0.05 level (df = 30). Conclusions: Results of the present study prove tropolone derivative to be prospective candidates for anti-metastatic drug development. Further investigations are needed to reveal the exact mechanisms of tropolones anti-migratory activity.

Discovery of a covalent inhibitor of heat shock protein 90 with antitumor activity that blocks the co-chaperone binding via C-terminal modification

Heat shock protein (Hsp90), a critical molecular chaperone that regulates the maturation of a large number of oncogenic client proteins, plays an essential role in the growth of neoplastic cells. Herein, DDO-6600 is identified to covalent modification of Cys598 on Hsp90 from in silico study and is verified by a series of biological assays. We demonstrated that DDO-6600 covalently bound to Cys598 on the Hsp90 C terminus and exhibited antiproliferative activities against multiple tumor cells without inhibiting ATPase activity. Further studies showed that DDO-6600 disrupted the interaction between Hsp90 and Cdc37, which induced the degradation of kinase client proteins in multiple tumor cell lines, promoted apoptosis, and inhibited cell motility. Our findings offer mechanic insights into the covalent modification of Hsp90 and provide an alternative strategy for the development of Hsp90 covalent regulators or chemical probes to explore the therapeutical potential of Hsp90.

PRR11 induces filopodia formation and promotes cell motility via recruiting ARP2/3 complex in non-small cell lung cancer cells

Filopodia, a finger-like structure and actin-rich plasma-membrane protrusion at the leading edge of the cell, has important roles in cell motility. However, the mechanisms of filopodia generation are not well-understood via the actin-related protein 2/3 (ARP2/3) complex in Non-Small Cell Lung Cancer (NSCLC) cells. We previously have demonstrated that PRR11 associates with the ARP2/3 complex to regulate cytoskeleton-nucleoskeleton assembly and chromatin remodeling. In this study, we further demonstrate that PRR11 involves in filopodia formation, focal adhesion turnover and cell motility through ARP2/3 complex. Cell phenotype assays revealed that the silencing of PRR11 increased cellular size and inhibited cell motility in NSCLC cells. Mechanistically, PRR11 recruited and co-localized with Arp2 at the membrane protrusion to promote filopodia formation but not lamellipodia formation. Notably, PRR11 mutant deletion of the proline-rich region 2 (amino acid residues 185–200) abrogated the effect of filopodia formation. In addition, PRR11-depletion inhibited filopodial actin filaments assembly and increased the level of active integrin β1 in the cell surface, whereas reduced the phosphorylation level of focal adhesion kinase (FAKY397) to repress focal adhesion turnover and cell motility in NSCLC cells. Taken together, our findings indicate that PRR11 has critical roles in controlling filopodia formation, focal adhesion turnover and cell motility by recruiting ARP2/3 complex, thus dysregualted expression of PRR11 potentially facilitates tumor metastasis in NSCLC cells.

DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer.

DUOX2 has been reported to highly express in several types of cancers. However, the prognostic significance and the biological function of DUOX2 expression with pancreatic cancer (PC) still remain unclear. The present study is aimed at investigating whether DUOX2 could act as a novel biomarker of prognosis and evaluating its effect on PC cell progression. The mRNA and protein expression of DUOX2 in PC cells and tissues were assessed by quantitative real-time PCR (RT-qPCR) and immunohistochemistry. The effect of DUOX2 expression on PC cell motility and proliferation was evaluated in vitro. The correlation between DUOX2 mRNA expression and clinicopathological features and its prognostic significance were analyzed according to the Gene Expression Profiling Interactive Analysis (GEPIA) website based on The Cancer Genome Atlas (TCGA) and the GTEx databases combined with our clinical information. According to bioinformatics analysis, we forecasted the upstream transcription factors (TFs) and microRNA (miRNA) regulatory mechanism of DUOX2 in PC. The expression of DUOX2 at transcriptional and protein level was dramatically increased in PC specimens when compared to adjacent nontumor specimens. Functionally, DUOX2 knockdown inhibited cell motility and proliferation activities. Our clinical data revealed that the patients had better postoperative overall survival (OS) with lower expression of DUOX2, which is consistent with GEPIA data. Multivariate analysis revealed that high DUOX2 expression was considered as an independent prognostic indicator for OS (P = 0.031). Based on Cistrome database, the top 5 TFs of each positively and negatively association with DUOX2 were predicted. hsa-miR-5193 and hsa-miR-1343-3p targeting DUOX2 were forecasted from TargetScan, miRDB, and DIANA-TarBase databases, which were negatively correlated with OS (P = 0.043 and P = 0.0088, respectively) and DUOX2 expression (P = 0.0093 and P = 0.0032, respectively) in PC from TCGA data. These findings suggest that DUOX2 acts as a promising predictive biomarker and an oncogene in PC, which could be a therapeutic target for PC.