Anchorage-dependent Cell Growth Research Articles

Seed Train Optimization in Microcarrier-Based Cell Culture Post In Situ Cell Detachment through Scale-Down Hybrid Modeling.

Microcarrier-based cell culture is a commonly used method to facilitate the growth of anchorage-dependent cells like MA 104 for antigen manufacturing. However, conventionally, static cell culture is employed for cell propagation before seeding the production bioreactor with microcarriers (MCs). This study demonstrates the effective replacement of the conventional method by serial subculturing on MCs with in situ cell detachment under optimal conditions in closed culture units. This study proves that MA 104 can be subcultured at least five times on Cytodex 1 MC without the need for separating cells and MC after cell harvest. Process parameters impacting cell growth were studied post in situ cell detachment in a scaled-down model. Optimization, using augmented Design of Experiments (DoE) combined with hybrid modeling, facilitated rapid screening of the design space for critical process parameters (CPPs). Optimized conditions included an inoculation density of >16 cells/bead, 3.5-4.5 g/L of Cytodex 1, and a controlled agitation speed, starting at Njs (minimum agitation speed) for the first day with a maximum increase of 25% thereafter. With these design spaces for CPPs, a cell density of 2.6 ± 0.5 × 106 cells/mL was achieved after five days. This refined bioprocess methodology offers a reliable and efficient approach for seed training in stirred tank reactors, which is particularly beneficial for viral vaccine production.

Downregulation of LAMB3 Altered the Carcinogenic Properties of Human Papillomavirus 16-Positive Cervical Cancer Cells.

Nearly all cervical cancer cases are caused by infection with high-risk human papillomavirus (HR-HPV) types. The mechanism of cervical cell transformation is related to the powerful action of viral oncoproteins and cellular gene alterations. Transcriptomic data from cervical cancer and normal cervical cells were utilized to identify upregulated genes and their associated pathways. The laminin subunit beta-3 (LAMB3) mRNAwas overexpressed in cervical cancer and was chosen for functional analysis. The LAMB3 was predominantly expressed in the extracellular region and the plasma membrane, which play a role in protein binding and cell adhesion molecule binding, leading to cell migration and tissue development. LAMB3 was found to be implicated in the pathway in cancer and the PI3K-AKT signaling pathway. LAMB3 knockdown decreased cell migration, invasion, anchorage-dependent and anchorage-independent cell growth and increased the number of apoptotic cells. These effects were linked to a decrease in protein levels involved in the PI3K-AKT signaling pathway and an increase in p53 protein. This study demonstrated that LAMB3 could promote cervical cancer cell migration, invasion and survival.

Phosphorylation of INF2 by AMPK promotes mitochondrial fission and oncogenic function in endometrial cancer

Mitochondria are highly dynamic organelles capable of altering their sizes and shapes to maintain metabolic balance through coordinated fission and fusion processes. In various cancer types, mitochondrial hyperfragmentation has been frequently observed, contributing to the progression of cancer toward metastasis. Inverted formin 2 (INF2), which resides in the endoplasmic reticulum (ER), has been found to accelerate actin polymerization and drive mitochondrial fission. In this study, we demonstrate that INF2 expression is significantly upregulated in endometrial cancer (EC) and is associated with a poor prognosis in EC patients. INF2 promotes anchorage-dependent and independent EC cell growth in part by facilitating mitochondrial fission. Furthermore, in conditions of energy stress, AMP-activated protein kinase (AMPK) phosphorylates INF2 at Ser1077, leading to increased localization of INF2 to the ER and enhanced recruitment of the dynamin-related protein 1 (DRP1) to mitochondria. This AMPK-mediated phosphorylation of INF2 at Ser1077 facilitates mitochondrial division and promotes EC cell growth. Pathological examination using immunohistochemical analyses revealed a positive correlation between AMPK activity and phosphorylated INF2 (Ser1077) in EC specimens. Collectively, our findings uncover novel molecular mechanisms involving the AMPK-INF2 axis, which regulates mitochondrial dynamics and malignant cell growth in EC.

Continuous topical application of microencapsulated recombinant human epidermal growth factor does not promote the progression of established melanoma in animals

Microencapsulated epidermal growth factor (EGF) device has been applied topically for the management of several types of wounds to accelerate wound healing and prevent scar formation. However, it remains unclear whether such EGF device induced neoplastic transformation in the skin. In this study, we exploited a well-established murine B16-F10 melanoma model, coupled with MTT viability and colony formation assays, to investigate the influence of microencapsulated recombinant EGF (Me-EGF; brand name NewEpi®) and its ingredients on the tumorigenicity of skin cancer cells in vitro and in vivo. The results indicated that Me-EGF did not stimulate the viability nor the anchorage-dependent growth of B16-F10 melanoma cells. Western blot analysis showed that Me-EGF treatment increased the total and phosphorylated EGFR expression without affecting the HER2 expression in B16-F10 melanoma cells. In mice bearing established B16-F10 melanoma, continuous application of Me-EGF for 14 days did not enhance the melanoma tumor burden compared with control groups. Immunohistochemical analysis also revealed the similar expression of proliferative index Ki-67 between Me-EGF-treated melanoma and other groups. Altogether, these results suggest that the application of Me-EGF device did not promote the oncogenic potential of B16-F10 melanoma in vitro and in vivo.

Cytophilic Agarose-Epoxide-Amine Cryogels Engineered with Granulated Microstructures.

Inherent cytophobicity of agarose limits its direct use for the growth of anchorage-dependent cells. Here, we report a simple strategy allowing the development of agarose-based hydrogels entailed with both cytophilicity and microstructured morphology. Through the reaction of water-soluble 1,4-butanediol diglycidyl ether (BDDE) with trifunctional polyetheramine Jeffamine T403 in agarose solution followed by cryogelation of the mixtures, a series of macroporous agarose-epoxide-amine cryogels were prepared readily. Results from fluorescent labeling and energy-dispersive X-ray elemental mapping showed the formation of granulated microstructures in the cryogels. Such features closely correlated to the phase separation of BDDE-T403 polymers within the agarose matrix. Cytophilicity of the microstructured cryogels due to the integrated amine moieties was demonstrated through the adhesion of fibroblasts. Functional enrichment of the cryogels was further highlighted by leveraging the granulates as micro-reservoirs for polyphenol proanthocyanidin to enable antioxidation and protection of fibroblasts from H2O2-induced cytotoxic effect in vitro.

Conoidin A Abrogates Growth of Anchorage-Dependent and Anchorage-Independent SKOV3 Ovarian Cancer Cells

Ovarian cancer is one of the leading causes of cancer death in the United States with only 49% of women surviving 5 years after initial diagnosis. Poor screening methods, chemoresistance, and its high metastatic capacity make this disease difficult to treat. Thus, improved treatment methodologies are necessary to improve survival rates and quality of life for ovarian cancer patients. Conoidin A is a water-soluble covalent inhibitor of peroxiredoxin 1 (PRDX1) and peroxiredoxin 2 (PRDX2). While its effects have been primarily studied on parasites, recent studies also begin to elucidate the impacts of conoidin A in other human health conditions, including anticancer effects. However, the anticancer effects and potential utility of conoidin A as a therapeutic, specifically in ovarian cancer, has yet to be investigated. Here, we report that conoidin A eliminates growth of anchorage-dependent and anchorage-independent SKOV3 cells. Furthermore, analyses of clinical samples show increased expression of PRDX1 and PRDX2 in ovarian cancerous tissue, which are the targets of conoidin A. These data identify conoidin A as a potential therapeutic for early and late-stage ovarian cancer and warrants further investigation. (First Online: December 31, 2022)

Slit2 signaling stimulates Ewing sarcoma growth.

Ewing sarcoma is a cancer of bone and soft tissue in children driven by EWS::ETS fusion, most commonly EWS::FLI1. Because current cytotoxic chemotherapies are not improving the survival of those with metastatic or recurrent Ewing sarcoma cases, there is a need for novel and more effective targeted therapies. While EWS::FLI1 is the major driver of Ewing sarcoma, EWS::FLI1 has been difficult to target. A promising alternative approach is to identify and target the molecular vulnerabilities created by EWS::FLI1. Here we report that EWS::FLI1 induces the expression of Slit2, the ligand of Roundabout (Robo) receptors implicated in axon guidance and multiple other developmental processes. EWS::FLI1 binds to the Slit2 gene promoter and stimulates the expression of Slit2. Slit2 inactivates cdc42 and stabilizes the BAF chromatin remodeling complexes, enhancing EWS::FLI1 transcriptional output. Silencing of Slit2 strongly inhibited anchorage-dependent and anchorage-independent growth of Ewing sarcoma cells. Silencing of Slit2 receptors, Robo1 and Robo2, inhibited Ewing sarcoma growth as well. These results uncover a new role for Slit2 signaling in stimulating Ewing sarcoma growth and suggest that this pathway can be targeted therapeutically.

Microgels-on-macrogel: A simple cytophilic surface makeover of soft agarose substrates

Microgel-mediated surface modification has shown great promises for a variety of metallic and non-metallic substrates. Yet, despite its compelling merits, this approach is less implemented on soft hydrogel substrates. Here, using the well-known bioinert agarose hydrogel as an example, we highlight a microgels-on-macrogel strategy that readily confers cytophilicity to the agarose surface toward anchorage-dependent cells. Specifically, we selected glycerol diglycidyl ether to tailor design polyetheramine-bisepoxide-based cationic microgels with more prominent ether alcohol features for enhanced chemical compatibility with agarose. Through a simple drop casting method, concurrent modifications of chemical, morphological and mechanical properties of the surface of agarose gel were then achieved with these microgels bound to the surface in a non-covalent yet robust manner. With the mere introduction of the cationic microgels, not only was the non-adhesive agarose surface effectively transformed to be cytophilic shown by the favorable responses from the in vitro culture of MC3T3-E1 cells, but also was hydrophobic reservoir function integrated conveniently. The demonstration of its feasibility and versatility warrants continued research of this straightforward microgels-on-macrogel strategy, which could be of value particularly for the development of novel biointerfaces. • Non-vinylic cationic microgels with prominent ether alcohol features readily prepared in water. • Facile yet robust surface modification of agarose achieved by sessile drop casting of the microgels. • Concurrent changes in agarose surface chemistry, morphology and stiffness mediated by the microgels. • Adhesion and growth of anchorage-dependent cells enabled via surface-bound microgels. • Hydrophobic depot function readily integrated into the surface of a hydrophilic gel substrate.

Hybrid Pharmacophore- and Structure-Based Virtual Screening Pipeline to Identify Novel EGFR Inhibitors That Suppress Non-Small Cell Lung Cancer Cell Growth.

Dysregulated epidermal growth factor receptor (EGFR) expression is frequently observed in non-small cell lung cancer (NSCLC) growth and metastasis. Despite recent successes in the development of tyrosine kinase inhibitors (TKIs), inevitable resistance to TKIs has led to urgent calls for novel EGFR inhibitors. Herein, we report a rational workflow used to identify novel EGFR-TKIs by combining hybrid ligand- and structure-based pharmacophore models. Three types of models were developed in this workflow, including 3D QSAR-, common feature-, and structure-based EGFR-TK domain-containing pharmacophores. A National Cancer Institute (NCI) compound dataset was adopted for multiple-stage pharmacophore-based virtual screening (PBVS) of various pharmacophore models. The six top-scoring compounds were identified through the PBVS pipeline coupled with molecular docking. Among these compounds, NSC609077 exerted a significant inhibitory effect on EGFR activity in gefitinib-resistant H1975 cells, as determined by an enzyme-linked immunosorbent assay (ELISA). Further investigations showed that NSC609077 inhibited the anchorage-dependent growth and migration of lung cancer cells. Furthermore, NSC609077 exerted a suppressive effect on the EGFR/PI3K/AKT pathway in H1975 cells. In conclusion, these findings suggest that hybrid virtual screening may accelerate the development of targeted drugs for lung cancer treatment.

Abstract P4-02-14: Breast cancer cell/adipocyte crosstalk in obesity hampers the efficacy of tamoxifen

Abstract Despite the improvements in the outcomes of breast cancer patients following the implementation of endocrine therapy into daily oncology practice, the development of ‘de novo’ or acquired resistance to these treatments has become a major limitation. To date, several hallmarks of hormonal resistance have been proposed, including ERα loss or mutations, activation of growth factor-mediated signaling pathways, and alterations of key cell cycle checkpoints. More recently, it was proven that a reduced response to these therapeutic regimens may rely not only on tumor-cell intrinsic factors, but it is also dependent on the surrounding microenvironment and on the host characteristics. Indeed, clinical studies have suggested that obesity, in addition to promoting breast cancer development and progression, represents a heavyweight player driving breast cancer endocrine resistance. However, the influence of adipocytes and their secretome on the efficacy of hormone therapy has not properly investigated. In a model of co-culture, we demonstrated that the anti-proliferative effects of Tamoxifen on anchorage-dependent and anchorage-independent growth of MCF-7 breast cancer cells were counteracted by conditioned media (CM) released by fully differentiated 3T3-L1 cells (3T3-L1 A), a widely used 'in vitro' model of white adipocytes. Moreover, pre-incubation of MCF-7 cells with adipocyte-derived CM reduced their sensitivity to the inhibitory effects of Tamoxifen on motility, as evidenced by wound-healing and transmigration assays. Interestingly, the growth and the motility of Tamoxifen-resistant breast cancer cells were dramatically increased in the presence of adipocyte-derived CM, further suggesting a supportive role of adipocyte secretome in the development and progression of endocrine-resistant phenotypes. Quantitative transcriptome profiling of Tamoxifen-resistant cells revealed 917 differentially expressed genes (FDR ≤ 0.05), of which 243 were upregulated and 180 were downregulated in response to adipocyte-CM. Among these major modifications, the most significant down-regulated gene was Thioredoxin-interacting protein (TXNIP, 5-fold), a tumor suppressor participating in the metabolic reprogramming and oxidative stress in various solid cancers. These results well correlated with data obtained in retrospective analyses showing that low TXNIP expression in breast cancer patients was associated with a statistically significant poorer recurrence-free (RFS), overall (OS), post-progression (PPS) and distant metastasis-free (DMFS) survival compared to high TXNIP-expressing patients. This relevant discrimination is also achieved in tamoxifen-treated patients, highlighting a role of TXNIP for identifying patients at high risk of progression. These findings bring insights on adipocytes and mammary cancer cell interactions during Tamoxifen therapy, particularly in overweight/obese people and add evidence to advocating obesity as a parameter to be considered when identifying treatments for patients with ER-positive breast cancer. Citation Format: Amanda Caruso, Luca Gelsomino, Giusi La Camera, Cinzia Giordano, Salvatore Panza, Daniela Bonofiglio, Stefania Catalano, Sebastiano Andò, Ines Barone. Breast cancer cell/adipocyte crosstalk in obesity hampers the efficacy of tamoxifen [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-14.

Abstract 1230: High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype

Abstract Signal Transducer and Activator of Transcription (STAT)3 is a member of the STAT family of transcription factors that is frequently aberrantly-activated in breast cancer and many other cancers. STAT3 represents a valid target for the development of novel anticancer therapeutics. However, it has posed significant challenges to the discovery efforts to identify potent and suitable small molecule inhibitors. As a result, there is currently no anti-STAT3 drugs in clinical application to realize the potential clinical benefits of inhibiting abnormal STAT3 function. Herein is a new series of (R)-azetidine-2-carboxamide-based small molecules, exemplified by H098, H127, H152, H169, H174, H182, and H203, which selectively inhibit STAT3 DNA-binding activity, with in vitro potency (IC50) in the range of 0.28-0.66 μM, compared to potencies greater than 18 µM against the activities of the family members, STAT1 and STAT5. Equally notable is the high affinity binding to STAT3, as shown by KD of 1 pM and 3.7 nM for H169 and H174, respectively. Treatment of human triple-negative breast cancer (TNBC) cells with H182 or H169 inhibited constitutive STAT3 phosphorylation (pY705STAT3) and DNA-binding activity in both time- and dose-dependent manner. Furthermore, both inhibitors potently inhibited anchorage-dependent and independent growth, colony survival, and migration of TNBC cells in vitro, with potencies, EC50 of 1.0-1.9 μM, and the compounds also induced apoptosis of same cells. Additionally, the expression of STAT3 target genes, including c-Myc, VEGF, and survivin, were suppressed in the compound-treated TNBC cells subsequent to the inhibition of constitutive STAT3 activation, thereby validating the inhibition of constitutively-active STAT3 and its downstream targets as being responsible for the antitumor cell responses. Moreover, H169 and docetaxel combinatorial treatment led to enhanced suppression of TNBC cell viability. By contrast, treatments with the new compounds had minimal effects on the induction of STAT1, Janus kinase 2, epidermal growth factor receptor, extracellular signal-regulated kinases (Erk1/2), Src, Shc phosphatase, or Akt in TNBC cells, and they were relatively weak on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively-active STAT3. Significantly, H182 delivered at 5-20 mg/kg via oral gavage or intra-peritoneal route inhibited growth of TNBC tumor xenograft in mice, with no evidence of toxicity. These studies identify novel, sub-micromolar potent azetidine-based small molecule STAT3 inhibitors that have shown antitumor efficacy against human TNBC in xenograft models. H182 and H169 represent suitable novel chemical entities for developing new therapeutics against TNBC and other cancers that harbor constitutively-active STAT3. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Yinsong Zhu, Christine Brotherton-Pleiss, Wenzhen Fu, Felix Alonso-Valenteen, Simoun Mikhael, Lali Medina-Kauwe, Marcus Tius, James Turkson. High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype [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 1230.

2,6-DMBQ suppresses cell proliferation and migration via inhibiting mTOR/AKT and p38 MAPK signaling pathways in NSCLC cells

2,6-Dimethoxy-1,4-benzoquinone (2,6-DMBQ) is the major bioactive compound found in fermented wheat germ extract. Although fermented wheat germ extract has been reported to show anti-proliferative and anti-metabolic effects in various cancers, the anticancer potential and molecular mechanisms exerted by 2,6-DMBQ have not been investigated in non-small cell lung cancer (NSCLC) cells. Here, we report that 2,6-DMBQ suppresses NSCLC cell growth and migration through inhibiting activation of AKT and p38 MAPK. 2,6-DMBQ significantly suppressed anchorage-dependent and independent cell growth. Additionally, 2,6-DMBQ induced G2 phase cell cycle arrest through inhibiting the expression and phosphorylation of cyclin B1 and CDC2, respectively. Furthermore, 2,6-DMBQ strongly suppressed NSCLC cell migration through induction of E-cadherin expression. To determine the molecular mechanism(s) exerted by 2,6-DMBQ upon NSCLC cell lines, various signaling kinases were screened; the results indicate that 2,6-DMBQ strongly inhibits the phosphorylation of AKT and p38 MAPK. Additionally, the growth kinetics of cells treated with an AKT or p38 MAPK inhibitor in combination with 2,6-DMBQ indicate that 2,6-DMBQ suppresses NSCLC cell growth and migration through inhibition of AKT and p38 MAPK. Taken together, our results suggest that 2,6-DMBQ is a potential anticancer reagent against NSCLC cells and could be useful for treating lung cancer patients.

Expansion and characterization of mesenchymal stem cells and other anchorage-dependent cell types in fluoropolymer bags treated for adherent cell culture

Background & Aim Modern cell based therapies continue to gain momentum as demonstrated by the growing number of clinical trials and cell therapy commercialization centers. A growing shift towards closed manufacturing systems to replace conventional functionally open systems such as tissue culture polystyrene (TCPS) flasks has prompted the development of various oxygen-permeable cell culture bags made from materials such as fluorinated ethylene propylene (FEP). However, hydrophobic materials such as untreated TCPS or FEP do not support adequately the growth of anchorage-dependent cells. For this reason, most TCPS vessels sold for adherent culture undergo plasma or liquid based surface treatments which increase surface wettability and cell adhesion. Unlike TCPS, the effect of FEP vessels treated for adherent culture on cell adhesion is poorly documented. This is a major obstacle for the production of anchorage-dependent cell based therapies in a closed setting. The objective of this study was to investigate the impact of the treated form of FEP on adherent cell cultures. Methods, Results & Conclusion Different types of anchorage-dependent cells, including mesenchymal stem cells (MSCs), mouse insulinoma 6 (MIN6) cells and human embryonic kidney (HEK) cells, were cultured in treated-FEP (VueLife® AC Series) bags and their biological properties were analyzed in comparison to cells seeded on untreated-FEP (VueLife® C Series) and/or treated-TCPS (Nunclon™ Delta-treated T-flasks). Light microscopy revealed that surface treatment enhanced adhesion on FEP, but final adherent cell yields after expansion remained lower than on treated-TCPS. Additionally, we observed that surface treatment of FEP did not affect the differentiation status of expanded MSCs or MIN6 populations. As expected, the removal of proteins from the medium or integrin inhibition reduced cell adhesion. Overall, our results demonstrate that a wide range of anchorage-dependent cells, including primary cells (MSCs) and immortalized cell lines (anchorage-dependent HEK cells, MIN6) can be successfully expanded in VueLife® AC bags. As an increasing number of adhesion cell based therapies are ready to enter the clinic, there is a pressing need to find the appropriate cell culture vessels and materials for better clinical outcomes.

Obesity and Breast Cancer: Unraveling the Role of Adipocyte‐Derived Exosomes

Excessive adiposity strongly influences risk, prognosis and progression of breast cancer. The revised concept of adipose tissue from an inert depot for body energy to an endocrine and immunologically active organ placed growing interest on the potential role of adipocyte secreted soluble factors and vesicles, particularly exosomes, as important players in cancer biology. In the last decades, several reports demonstrated that exosomes, small membrane‐derived vesicles, are able to modify target cell phenotype, by transferring bioactive molecules such as proteins, mRNAs, microRNAs, DNAs, lipids and transcriptional factors. However, whether adipose tissue‐derived exosomes, acting as a mode of cell‐to‐cell communication, might have a role in mediating the adverse effects of obesity on breast cancer is still not completely understood.Here, we evaluated the effects of exosomes derived from differentiated 3T3‐L1 adipocytes (3T3‐L1 A), a well recognized ‘in vitro’ model of white adipocytes, on breast cancer phenotype, using as experimental models both estrogen receptor α (ERα)‐positive MCF‐7 and triple negative MDA‐MB‐231 breast cancer cells.Exosomes from 3T3‐L1 A were isolated by sequential ultracentrifugation method, and characterized by Nanoparticle Tracking Analysis, Transmission Electron Microscopy and Immunoblotting analyses. 3T3‐L1 A‐ derived exosome uptake in both breast cancer cell lines was tested by monitoring internalization of exosomes labeled with PKH‐26. Treatment of MCF‐7 and MDA‐MB‐231 breast cancer cells with adipocyte‐derived exosomes did not modify anchorage‐dependent cell growth, while it significantly increased the ability of cells to form colonies in anchorage‐independent soft agar growth assays. In addition, we found that both breast cancer cells treated with adipocyte‐derived exosomes displayed increased migration and invasion capabilities as evidenced by wound healing, and Boyden Chamber migration and invasion assays. Accordingly, adipocyte‐derived exosomes significantly induce the expression of genes involved in Epithelial to Mesenchymal Transition (EMT) in both cell lines. Since migratory potential and EMT represent important features for breast cancer stem cells (BCSCs), we also explored the effect of 3T3L‐1 A‐derived exosomes on MCF‐7 and MDA‐MB‐231 BCSC activity. Interestingly, we found that adipocyte‐derived exosomes were able to increase Mammosphere Forming Efficiency and Self Renewal in breast cancer cells. In conclusion, our data highlight how adipocyte‐derived exosomes may contribute to a more aggressive breast cancer phenotype, providing novel insight in the link between obesity and breast cancer. This may open a new molecular window for preventive and therapeutic strategies to fight breast cancer in obese patients.Support or Funding InformationThis research was funded by Fondazione AIRC: IG #11595 to SA and IG #21414 to SC, and PRIN2017 #2017EKMFTN to SC; PRIN2017 #2017WNKSLR to IB

Nanosized iron-substituted hydroxyapatites

Introduction of iron ions into hydroxyapatites imparts some useful properties to ceramic materials. We created a porous ceramic material from nanosized iron-substituted HA and studied its physico-chemical and biological properties. It is shown that all the iron ions introduced in the course of synthesis enter into the composition of HA and are characterized by the oxidation state +3. The size of crystals after thermal treatment at 9000 C does not exceed 100 nm, the crystals’ shape is close to a sphere. Porous materials from nanosized iron-substituted hydroxyapatites support adhesion and growth of anchorage-dependent cells of mammals.

Scutellarin Suppresses Patient-Derived Xenograft Tumor Growth by Directly Targeting AKT in Esophageal Squamous Cell Carcinoma.

Scutellarin is a flavonoid compound that is found in Scutellaria barbata It has been reported to exhibit anticancer and anti-inflammation activities. However, the anticancer properties of scutellarin and its molecular targets have not been investigated in esophageal squamous cell carcinoma (ESCC). In the current study, we report that scutellarin is a potential AKT inhibitor that suppresses patient-derived xenograft ESCC tumor growth. To identify possible molecular targets of scutellarin, potential candidate proteins were screened by an in vitro kinase assay and Western blotting. We found that scutellarin directly binds to the AKT1/2 proteins and inhibits activities of AKT1/2 in vitro The AKT protein is activated in ESCC tissues and knockdown of AKT significantly suppresses growth of ESCC cells. Scutellarin significantly inhibits anchorage-dependent and independent cell growth and induces G2 phase cell-cycle arrest in ESCC cells. The inhibition of cell growth by scutellarin is dependent on the expression of the AKT protein. Notably, scutellarin strongly suppresses patient-derived xenograft ESCC tumor growth in an in vivo mouse model. Taken together, our data suggest that scutellarin is a novel AKT inhibitor that may prevent progression of ESCC.

Pharmacological inhibition of ABCC3 slows tumour progression in animal models of pancreatic cancer

BackgroundPancreatic Ductal Adenocarcinoma (PDAC) is an aggressive and lethal disease, lacking effective therapeutic approaches. Available therapies only marginally prolong patient survival and are frequently coupled with severe adverse events. It is therefore pivotal to investigate novel and safe pharmacological approaches. We have recently identified the ABC transporter, ABCC3, whose expression is dependent on mutation of TP53, as a novel target in PDAC. ABCC3-mediated regulation of PDAC cell proliferation and tumour growth in vivo was demonstrated and was shown to be conferred by upregulation of STAT3 signalling and regulation of apoptosis.MethodsTo verify the potential of ABCC3 as a pharmacological target, a small molecule inhibitor of ABCC3, referred to here as MCI-715, was designed. In vitro assays were performed to assess the effects of ABCC3 inhibition on anchorage-dependent and anchorage-independent PDAC cell growth. The impact of ABCC3 inhibition on specific signalling pathways was verified by Western blotting. The potential of targeting ABCC3 with MCI-715 to counteract PDAC progression was additionally tested in several animal models of PDAC, including xenograft mouse models and transgenic mouse model of PDAC.ResultsUsing both mouse models and human cell lines of PDAC, we show that the pharmacological inhibition of ABCC3 significantly decreased PDAC cell proliferation and clonal expansion in vitro and in vivo, remarkably slowing tumour growth in mice xenografts and patient-derived xenografts and increasing the survival rate in a transgenic mouse model. Furthermore, we show that stromal cells in pancreatic tumours, which actively participate in PDAC progression, are enriched for ABCC3, and that its inhibition may contribute to stroma reprogramming.ConclusionsOur results indicate that ABCC3 inhibition with MCI-715 demonstrated strong antitumor activity and is well tolerated, which leads us to conclude that ABCC3 inhibition is a novel and promising therapeutic strategy for a considerable cohort of patients with pancreatic cancer.

Abstract LB-287: Combination therapies with CDK4/6 inhibitors to treat KRAS-mutant pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) patients have a dismal five-year survival rate of just eight percent in the advanced metastatic setting. Outcomes with standard chemotherapy regimens are less than ideal; therefore, the development of targeted therapies for the treatment of PDAC is a significant unmet clinical need. The two most frequent genetic events in PDAC (mutational activation of KRAS and loss of the tumor suppressor CDKN2A) converge on activation of the kinases CDK4 and CDK6, which promote G1 cell cycle progression. While clinically relevant agents directly targeting KRAS in PDAC remain elusive, pharmacologic restoration of CDKN2A function by inhibition of CDK4/6 may be an effective anti-KRAS therapeutic strategy. Supporting this hypothesis, we found that CDK4/6 inhibitors (e.g., palbociclib, abemaciclib, lerociclib) elicited single-agent activity in a subset of tested PDAC cell lines. However, using Reverse Phase Protein Array (RPPA), we observed widespread compensatory changes that overcome CDK4/6 inhibition, including increased anti-apoptotic protein expression, PI3K-mTOR and ERK MAPK signaling. Concurrent treatment with the potent and selective ERK1/2 inhibitor (ERKi) SCH772984 reversed resistance and increased sensitivity to palbociclib. This combination caused synergistic reduction in anchorage-dependent cell growth, and increased apoptosis, G1 arrest, and senescence. In an organoid model of pancreatic cancer, cell viability was synergistically reduced and caspase activation enhanced. We have also evaluated the combination of ERKi with palbociclib or the clinical-stage CDK4/6 inhibitor lerociclib (G1T38) in mouse models of PDAC, supporting our initiation of a phase Ib clinical trial of the ERK inhibitor ulixertinib/BVD-523 in combination with palbociclib in advanced metastatic pancreatic cancer (NCT03454035). Next, we sought to identify additional genes that regulate sensitivity to CDK4/6 inhibition. Using a CRISPR/Cas9 loss-of-function screen, we individually silenced expression of 2500 genes from the “druggable genome” in combination with palbociclib in six KRAS-mutant pancreatic and two KRAS-mutant colorectal cancer cell lines. We identified a wide array of genes that enhanced growth suppression in combination, centered around diverse signaling nodes including PI3K-AKT-mTOR signaling, cell cycle regulation and mitosis, SRC family kinase signaling, cell metabolism and biosynthesis, chromosome regulation and maintenance, and DNA damage & repair pathways, suggesting novel small molecule combinations to overcome de novo or acquired CDK4/6 inhibitor resistance in the clinic. We also identified genes whose loss imparts a survival advantage, suggesting possible resistance mechanisms to single-agent CDK4/6 inhibition. In total, these data suggest that CDK4/6 inhibitors alone, or in combination, may benefit PDAC patients clinically. Citation Format: Craig M. Goodwin, Sehrish Javaid, Andrew M. Waters, Bjoern Papke, Runying Yang, Mariaelena Pierobon, Daniel M. Freed, Patrick J. Roberts, Adrienne D. Cox, Kris C. Wood, Emanuel F. Petricoin, Autumn J. McRee, Channing J. Der. Combination therapies with CDK4/6 inhibitors to treat KRAS-mutant pancreatic cancer [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 LB-287.

Lapachol is a novel ribosomal protein S6 kinase 2 inhibitor that suppresses growth and induces intrinsic apoptosis in esophageal squamous cell carcinoma cells.

Lapachol is a 1,4-naphthoquinone that is isolated from the Bignoniaceae family. It has been reported to exert anti-inflammatory, antibacterial, and anticancer activities. However, the anticancer activity of lapachol and its molecular mechanisms against esophageal squamous cell carcinoma (ESCC) cells have not been fully investigated. Herein, we report that lapachol is a novel ribosomal protein S6 kinase 2 (RSK2) inhibitor that suppresses growth and induces intrinsic apoptosis in ESCC cells. We found that lapachol strongly attenuates downstream signaling molecules of RSK2 in ESCC cells and also directly inhibits RSK2 activity in vitro. The RSK protein is highly activated in ESCC cells and knockdown of RSK2 significantly suppresses anchorage-dependent and anchorage-independent growth of ESCC cells. Additionally, lapachol inhibits anchorage-dependent and anchorage-independent growth of ESCC cells, and the inhibition of cell growth by lapachol is dependent on the expression of RSK2. We also found that lapachol induces mitochondria-mediated cellular apoptosis by activating caspases-3, -7, and PARP, inducing the expression of cytochrome c and BAX by inhibiting downstream molecules of RSK2. Overall, lapachol is a potent RSK2 inhibitor that might be used for chemotherapy against ESCC.

Ovarian Cancer Relies on Glucose Transporter 1 to Fuel Glycolysis and Growth: Anti-Tumor Activity of BAY-876.

The recent progresses in understanding of cancer glycolytic phenotype have offered new strategies to manage ovarian cancer and other malignancies. However, therapeutic targeting of glycolysis to treat cancer remains unsuccessful due to complex mechanisms of tumor glycolysis and the lack of selective, potent and safe glycolytic inhibitors. Recently, BAY-876 was identified as a new-generation inhibitor of glucose transporter 1 (GLUT1), a GLUT isoform commonly overexpressed but functionally poorly defined in ovarian cancer. Notably, BAY-876 has not been evaluated in any cell or preclinical animal models since its discovery. We herein took advantage of BAY-876 and molecular approaches to study GLUT1 regulation, targetability, and functional relevance to cancer glycolysis. The anti-tumor activity of BAY-876 was evaluated with ovarian cancer cell line- and patient-derived xenograft (PDX) models. Our results show that inhibition of GLUT1 is sufficient to block basal and stress-regulated glycolysis, and anchorage-dependent and independent growth of ovarian cancer cells. BAY-876 dramatically inhibits tumorigenicity of both cell line-derived xenografts and PDXs. These studies provide direct evidence that GLUT1 is causally linked to the glycolytic phenotype in ovarian cancer. BAY-876 is a potent blocker of GLUT1 activity, glycolytic metabolism and ovarian cancer growth, holding promise as a novel glycolysis-targeted anti-cancer agent.