scholarly journals IFN-λ Modulates the Migratory Capacity of Canine Mammary Tumor Cells via Regulation of the Expression of Matrix Metalloproteinases and Their Inhibitors

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 999
Author(s):  
Rafał Pingwara ◽  
Daria Kosmala ◽  
Natalia Woźniak ◽  
Arkadiusz Orzechowski ◽  
Joanna Mucha
Keyword(s):  
Matrix Metalloproteinases ◽  
Cell Lines ◽  
Mammary Tumor ◽  
Tumor Development ◽  
Annexin V ◽  
Scratch Test ◽  
Metastatic Potential ◽  
Tumor Type ◽  
Canine Mammary Tumor

Interactions between neoplastic and immune cells taking place in tumors drive cancer regulatory mechanisms both in humans and animals. IFN-λ, a potent antiviral factor, is also secreted in the tumor; however, its role in tumor development is still unclear. In our study, we investigate the influence of IFN-λ on the canine mammary tumor (CMT) cell survival and their metastatic potential in vitro. First, we examined, by Western blot, the expression of the IFN-λ receptor complex in three CMT cell lines (P114, CMT-U27 and CMT-U309). We showed that only two cell lines (P114 and CMT-U27) express both (IL-28RA and IL-10Rb) receptor subunits and respond to IFN-λ treatment by STAT phosphorylation and the expression of interferon-stimulated genes. Using MTT, crystal violet and annexin-V assays, we showed a minimal role of IFN-λ in CMT viability. However, IFN-λ administration had a contradictory effect on cell migration in the scratch test, namely, it increased P114 and decreased CMT-U27 motility. Moreover, we demonstrated that this process is related to the expression of extracellular matrix metalloproteinases and their inhibitors; furthermore, it is independent of Akt and ERK signaling pathways. To conclude, we showed that IFN-λ activity is reliant on the expression of two receptor subunits and tumor type, but further investigations are needed.

scholarly journals Establishment of a novel human CIC-DUX4 sarcoma cell line, Kitra-SRS, with autocrine IGF-1R activation and metastatic potential to the lungs

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sho Nakai ◽  
Shutaro Yamada ◽  
Hidetatsu Outani ◽  
Takaaki Nakai ◽  
Naohiro Yasuda ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Fusion Gene ◽  
Primary Tumour ◽  
Chromosomal Rearrangements ◽  
Basic Research ◽  
Metastatic Potential ◽  
Original Tumour

Abstract Approximately 60–70% of EWSR1-negative small blue round cell sarcomas harbour a rearrangement of CIC, most commonly CIC-DUX4. CIC-DUX4 sarcoma (CDS) is an aggressive and often fatal high-grade sarcoma appearing predominantly in children and young adults. Although cell lines and their xenograft models are essential tools for basic research and development of antitumour drugs, few cell lines currently exist for CDS. We successfully established a novel human CDS cell line designated Kitra-SRS and developed orthotopic tumour xenografts in nude mice. The CIC-DUX4 fusion gene in Kitra-SRS cells was generated by t(12;19) complex chromosomal rearrangements with an insertion of a chromosome segment including a DUX4 pseudogene component. Kitra-SRS xenografts were histologically similar to the original tumour and exhibited metastatic potential to the lungs. Kitra-SRS cells displayed autocrine activation of the insulin-like growth factor 1 (IGF-1)/IGF-1 receptor (IGF-1R) pathway. Accordingly, treatment with the IGF-1R inhibitor, linsitinib, attenuated Kitra-SRS cell growth and IGF-1-induced activation of IGF-1R/AKT signalling both in vitro and in vivo. Furthermore, upon screening 1134 FDA-approved drugs, the responses of Kitra-SRS cells to anticancer drugs appeared to reflect those of the primary tumour. Our model will be a useful modality for investigating the molecular pathology and therapy of CDS.

scholarly journals The Comprehensive Roles of ATRANORIN, A Secondary Metabolite from the Antarctic Lichen Stereocaulon caespitosum, in HCC Tumorigenesis

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1414 ◽  
Author(s):  
Young-Jun Jeon ◽  
Sanghee Kim ◽  
Ji Hee Kim ◽  
Ui Joung Youn ◽  
Sung-Suk Suh
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Genetic Diseases ◽  
Annexin V ◽  
Cancer Therapeutics ◽  
Metastatic Potential ◽  
Anti Cancer ◽  
Experimental Approaches ◽  
The Antarctic ◽  
Death Determination

Hepatocellular carcinoma (HCC) is one of the most deadly genetic diseases, but surprisingly chemotherapeutic approaches against HCC are only limited to a few targets. In particular, considering the difficulty of a chemotherapeutic drug development in terms of cost and time enforces searching for surrogates to minimize effort and maximize efficiency in anti-cancer therapy. In spite of the report that approximately one thousand lichen-derived metabolites have been isolated, the knowledge about their functions and consequences in cancer development is relatively limited. Moreover, one of the major second metabolites from lichens, Atranorin has never been studied in HCC. Regarding this, we comprehensively analyze the effect of Atranorin by employing representative HCC cell lines and experimental approaches. Cell proliferation and cell cycle analysis using the compound consistently show the inhibitory effects of Atranorin. Moreover, cell death determination using Annexin-V and (Propidium Iodide) PI staining suggests that it induces cell death through necrosis. Lastly, the metastatic potential of HCC cell lines is significantly inhibited by the drug. Taken these together, we claim a novel functional finding that Atranorin comprehensively suppresses HCC tumorigenesis and metastatic potential, which could provide an important basis for anti-cancer therapeutics.

scholarly journals PEG Graft Polymer Carriers of Antioxidants: In Vitro Evaluation for Transdermal Delivery

Pharmaceutics ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1178
Author(s):  
Justyna Odrobińska ◽  
Magdalena Skonieczna ◽  
Dorota Neugebauer
Keyword(s):  
Cell Lines ◽  
Graft Copolymers ◽  
Biological Response ◽  
Annexin V ◽  
Dermal Fibroblasts ◽  
In Vitro Tests ◽  
Side Chains ◽  
Skin Cell ◽  
Active Substances

The in vitro biochemical evaluation of the applicability of polymers carrying active substances (micelles and conjugates) was carried out. Previously designed amphiphilic graft copolymers with retinol or 4-n-butylresorcinol functionalized polymethacrylate backbone and poly(ethylene glycol) (PEG) side chains that included Janus-type heterografted copolymers containing both PEG and poly(ε-caprolactone) (PCL) side chains were applied as micellar carriers. The polymer self-assemblies were convenient to encapsulate arbutin (ARB) as the selected active substances. Moreover, the conjugates of PEG graft copolymers with ferulic acid (FA) or lipoic acid (LA) were also investigated. The permeability of released active substances through a membrane mimicking skin was evaluated by conducting transdermal tests in Franz diffusion cells. The biological response to new carriers with active substances was tested across cell lines, including normal human dermal fibroblasts (NHDF), human epidermal keratinocyte (HaCaT), as well as cancer melanoma (Me45) and metastatic human melanoma (451-Lu), for comparison. These polymer systems were safe and non-cytotoxic at the tested concentrations for healthy skin cell lines according to the MTT test. Cytometric evaluation of cell cycles as well as cell death defined by Annexin-V apoptosis assays and senescence tests showed no significant changes under action of the delivery systems, as compared to the control cells. In vitro tests confirmed the biochemical potential of these antioxidant carriers as beneficial components in cosmetic products, especially applied in the form of masks and eye pads.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

Improved Therapy of Multiple Myeloma by Combining Milatuzumab (Anti-CD74 Humanized mAb) and Bortezomib: In Vitro and In Vivo Results.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1176-1176
Author(s):  
Rhona Stein ◽  
David M. Goldenberg
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Leukemic Cells ◽  
Tumor Type ◽  
Term Survival ◽  
Significant Difference ◽  
Highly Effective ◽  
Ic50 Values

Abstract Background: The humanized anti-CD74 monoclonal antibody, milatuzumab (hLL1, or IMMU-115; Immunomedics, Inc, Morris Plains, NJ), is in clinical evaluation for therapy of multiple myeloma (MM) after preclinical evidence of activity in this tumor type (Stein et al, Blood2004;104:3705). Here we examine the ability of milatuzumab to increase the efficacy of drugs in MM cell lines. Methods: MTT cytotoxicity assays were performed on a panel of MM cell lines, including CAG, KMS11, KMS12-PE, and MC/CAR, to examine the effects of bortezomib, doxorubicin (dox), and dexamethasone (dex) alone and combined with milatuzumab or milatuzumab + crosslinking 2nd Ab (goat anti-human IgG, GAH). In vivo studies used a CAG-SCID mouse model of disseminated disease. Results: Without drugs, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects on the four MM cell lines. In combination studies, crosslinked milatuzumab produced significant reductions in the IC50 values of the anti-MM drugs. For example, in CAG, milatuzumab+GAH decreased the IC50 values 58%, 78%, and 98% for bortezomib, dox, and dex, respectively (P=0.0034, 0.0073, and 0.078, respectively). In vivo, milatuzumab at 100 μg/injection, 2x weekly for 4 weeks, starting 1 day after injection of CAG cells, more than doubled the median survival time (MST) from 42 days in untreated CAG-bearing SCID mice to 103 days. Combination therapy with milatuzumab and bortezomib or dox was compared to milatuzumab alone, with treatments initiated 5 days after injection of CAG cells. Bortezomib alone (1.0 mg/kg) increased MST from 33 to 44 days (P=0.0021 vs. untreated). Treatment with milatuzumab alone (100 μg/mouse) increased the MST to 73 days (P<0.0001 vs. untreated). When bortezomib and milatuzumab treatments were combined, the MST increased to 93 days (P=0.0441 vs. milatuzumab and P=0.0065 vs. bortezomib). Thus, the combination of milatuzumab and bortezomib increased survival significantly compared to either single treatment. Given alone, dox yielded little or no effect on survival compared with untreated animals, and there was no significant difference between milatuzumab monotherapy and milatuzumab plus doxorubicin in this model. In contrast, a milatuzumabdox immunoconjugate was found to be a highly effective therapeutic agent, with all mice achieving long-term survival. The inhibition of the NF-κB survival pathway of B-leukemic cells by milatuzumab supports its complementary effects when combined with drugs having different mechanisms of action, such as bortezomib. Conclusions: The therapeutic efficacies of bortezomib, dox, and dex are enhanced in vitro in MM cell lines when given in combination with milatuzumab. In vivo, milatuzumab alone or especially in combination with bortezomib is highly effective in MM. (Supported in part by USPHS grant P01CA103985 from the NCI, and grants from the Thomas and Agnes Carvel Foundation and the Walter and Louise Sutcliffe Foundation.)

Priming Effect with G-CSF Enhances In Vitro Apoptosis by Treatment with Ara-C and VP-16 in Leukemic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4333-4333
Author(s):  
Jun-ichi Kitagawa ◽  
Takeshi Hara ◽  
Hisashi Tsurumi ◽  
Nobuhiro Kanemura ◽  
Masahito Shimizu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Priming Effect ◽  
Low Dose ◽  
Combined Treatment ◽  
Annexin V ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Significant Difference

Abstract Introduction: We have recently reported that the effectiveness of low dose Ara-C, VP-16 and G-CSF (AVG therapy) for elderly AML patients who were ineligible for intensive chemotherapy (Hematol Oncol, in press). G-CSF has been reported to potentiate in vitro anti-leukemic effect of Ara-C. The mechanism of the potentiation is assumed to recruit quiescent G0 leukemic cells into cell cycle. We hypothesized that the enhanced cytotoxicity was due to the apoptosis by the effect of the priming of G-CSF, and the effect was depended on the cell cycle. In order to afford proof of this hypothesis, we assayed proliferation, apoptosis, and cell cycle in leukemic cell lines. Materials: Ara-C, VP-16, G-CSF was provided by Nippon Shinyaku, Nihonkayaku, Chugai pharmacy, respectively, Tokyo, Japan. 32D and HL-60 were obtained from RIKEN Bioresource Center Cell Bank (Ibaragi, Japan), Ba/F3 was generous gifts from Dr. Kume, Jichi medical school, Tochigi, Japan. Methods: 5 x 105/ml HL60, 32D and Ba/F3 were cultured with various concentrations of Ara-C and/or VP-16 in the presence or absence of G-CSF 50ng/ml for 3 days. At the end of the culture, cell proliferation and viability were determined by using the trypan blue. The Annexin V-binding capacity of treated cells was examined by flow cytometry using ANNEXIN V-FITC APOPTOSIS DETECTION KIT I purchased from BD Pharmingen™. Cell cycle analysis was done with BrdU Flow KIT purchased from BD Pharmingen™. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies, and the levels of cell-associated BrdU are then measured by flow cytometory. Result: Ara-C and VP-16 inhibited proliferation and decreased viability of leukemic cell lines dose-dependently. Half killing concentration (IC50) was redused in combination of Ara-C and VP-16 than Ara-C or VP-16 alone. In G-CSF dependent cell line (32D), IC50 was redeced in the presence of G-CSF than absence of G-CSF at G-CSF, and there was no significant difference between with and without G-CSF in G-CSF independent cell lines (HL-60, Ba/F3) (p<0.05). In combined treatment of low dose Ara-C (10−7M) and VP-16 (10−7M), the percentage of apoptotic cells were increased to 20.67% from 13.04% by addition of G-CSF in 32D, and there was no significant differencebetween with and without G-CSF in HL-60 and Ba/F3 (p<0.05). At combined treatment of low dose Ara-C and VP-16, the percentage of G0/G1 phase cells were decreased to 43.94% from 35.63% and S phase cells were increased to 29.50% from 24.05% in 32D by addition of G-CSF, and there was no significant difference between with and without G-CSF in HL-60 and Ba/F3 (p<0.05). Discussion: We first showed a combination effect of Ara-C and VP-16. Next we demonstrated that the potentiation of the cytotoxicity was mediated through the mechanism of apoptosis, and apoptosis played an important role for eradicating leukemic cells by low dose Ara-C and VP-16. And G-CSF recruited cells G0/G1 phase into S phase in G-CSF dependent cells by addition of G-CSF. These results suggest that priming effect of G-CSF significantly potentiate the cytotoxicity mediated by AVG chemotherapy. Conclusion: The priming effect of G-CSF might be admitted at least of a part in AML cells.

NPI-2358, a Novel Vascular Disrupting Agent Blocks Growth and Angiogenesis in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3842-3842
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Madhavi Bandi ◽  
Noopur Raje ◽  
Robert L Schlossman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Vascular Endothelial ◽  
Caspase 9 ◽  
Migration Assays ◽  
Induced Apoptosis ◽  
Rpmi 8226 ◽  
Anti Tumor Activity

Abstract Abstract 3842 Poster Board III-778 Background and Rationale Vascular disrupting agents (VDAs) act via selectively disrupting established tumor vasculature and have shown remarkable clinical success as anti-cancer therapies. NPI-2358 is a novel VDA with a distinct structure and mechanism of action from other available VDAs. NPI-2358 binds to the colchicine-binding site of beta-tubulin preventing polymerization and disrupting the cytoplasmic microtubule network, thereby causing loss of vascular endothelial cytoskeletal function, and inducing cytotoxicity in cancer cells. Here, we examined the anti-angiogenic and anti-tumor activity of NPI-2358 in multiple myeloma (MM) cells using both in vitro and in vivo model systems. Material and Methods We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from MM patients relapsing after prior anti-MM therapies. Cell viability/apoptosis assays were performed using MTT, trypan blue exclusion, and Annexin V/PI staining. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assays: Since human vascular endothelial cells (HUVECs) plated onto Matrigel differentiate and form capillary-like tube structures similar to in vivo neovascularization, this assay measures anti-angiogenic effects of drugs/agents. Migration assays were performed using transwell insert assays. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, Bax, pJNK and GAPDH. Statistical significance was determined using a Student t test. Results Treatment of MM.1S, RPMI-8226, MM.1R, INA-6, and KMS-12BM with NPI-2358 for 24h induces a dose-dependent significant (P < 0.005) decrease in viability of all cell lines (IC50 range: 5-8 nM; n=3). To determine whether NPI-2358-induced decrease in viability is due to apoptosis, MM cell lines were treated with NPI-2358 for 24h; harvested, and analyzed for apoptosis using Annexin V/PI staining. A significant increase in NPI-2358-induced apoptosis was observed in all MM cell lines (% Annexin V+/PI- apoptotic cells: MM.1S, 48 ± 2.3%; MM.1R, 46.6 ± 3.1%; RPMI-8226, 61.7 ± 4.5%; and INA-6, 59.9 ± 3.2%; P < 0.05; n=3). Importantly, NPI-2358 decreased viability of freshly isolated MM cells from patients (IC50 range: 3-7 nM; P < 0.005), without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for NPI-2358. Examination of in vitro angiogenesis using capillary-like tube structure formation assay showed that even low doses of NPI-2358 (7 nM treatment for 12h; IC50: 20 nM at 24h) significantly decreased tubule formation in HUVECs (70-80% decrease; P < 0.05). Transwell insert assays showed a marked reduction in serum-dependent migration of NPI-2358-treated MM cells (42 ± 2.1% inhibition in NPI-2358-treated vs. control; P < 0.05). NPI-2358 at the concentrations tested (5 nM for 12h) in the migration assays did not affect survival of MM cells (> 95% viable cells). A similar anti-migration activity of NPI-2358 was noted against HUVEC cells (48 ± 1.7% decrease in migration; P < 0.05). Mechanistic studies showed that NPI-2358-induced apoptosis was associated with activation of caspase-8, caspase-9, caspase-3 and PARP. Importantly, treatment of MM.1S cells with NPI-2358 (5 nM) triggered phosphorylation of c-Jun amino-terminal kinase (JNK), a classical stress response protein, without affecting Bcl-2 family members Bax and Bcl-2. Blockade of JNK using dominant negative strategy markedly abrogated NPI-2358-induced apoptosis. Conclusion Our preclinical data provide evidence for remarkable anti-angiogenic and anti-tumor activity of NPI-2358 against MM cells, without significant toxicity in normal cells. Ongoing studies are examining in vivo anti-MM activity of NPI-2358 in animal models. Importantly, a Phase-1 study of NPI-2358 as a single agent in patients with advanced malignancies (lung, prostrate and colon cancer) has already established a favorable pharmacokinetic, pharmacodynamic and safety profile; and, a Phase-2 study of the combination of NPI-2358 and docetaxel in non-small cell lung cancer showed encouraging safety, pharmacokinetic and activity data. These findings, coupled with our preclinical studies, provide the framework for the development of NPI-2358-based novel therapies to improve patient outcome in MM. Disclosures: Chauhan: Nereus Pharmaceuticals, Inc: Consultancy. Lloyd:Nereus Pharmaceuticals, In: Employment. Palladino:Nereus Pharmaceuticals, Inc: Employment. Anderson:Nereus Pharmaceuticals, Inc: Consultancy.

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