Journal: bioRxiv

Article Title: Lapatinib protects against epileptic seizures via halting glutathione peroxidase 4-dependent ferroptosis

doi: 10.1101/2020.05.28.120733

Figure Lengend Snippet: Lapatinib exerts neuroprotection against Glu- or erastin-induced cell death in HT22 neurons. (a) Indicated HT22 cells were treated with Glu (5 mM) or erastin (0.5 μM) for 8 h. Cell morphology was observed by phase-contrast microscopy after pretreatment with lapatinib at different concentrations (1.25, 2.5, 5 and 10 μM). Scale bar: 200 μm. (b) HT22 cells were pre-incubated with lapatinib (1.25, 2.5, 5 and 10 μM) for 2 h, followed with Glu or erastin treatment. Cell death rate was measured by PI (+)/Hoechst (+). (c, d) Lapatinib (10 μM) was put into the neuron cultures for different periods of time (before 6 h, 2 h, simultaneously or post 2 h) of Glu or erastin treatment. The percentage of cell death rate was measured by PI (+)/Hoechst (+). Scale bar: 200 μm. (e) Effects of different concentrations of lapatinib on HT22 cells. Scale bar: 200 μm. All results were shown as the mean ± SEM from three independent experiments, ***p < 0.001.

Article Snippet: Erastin, lapatinib, Fer-1, liproxstatin-1 (Lip-1) and Ras-selective lethal small molecule 3 (RSL3) were obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Microscopy, Incubation

Journal: bioRxiv

Article Title: Lapatinib protects against epileptic seizures via halting glutathione peroxidase 4-dependent ferroptosis

doi: 10.1101/2020.05.28.120733

Figure Lengend Snippet: Lapatinib prevented Glu- or erastin-induced neuronal death possibly by suppressing ferroptosis. (a-d) Detection of lipid ROS, 4-HNE and MDA content in the Glu- or erastin-induced HT22 cell injury model following lapatinib (10 μM) and Fer-1 (12.5 μM) pretreatment for 2 h. (e) RT-qPCR analysis of PTGS2 mRNA expression pretreated with or without lapatinib (10 μM) and Fer-1 (12.5 μM) in HT22 cells induced by Glu or erastin. (f, g) To observe and compare the effect between lapatinib and ferroptosis inhibitors co-intervention group and lapatinib intervention alone group on the Glu- or erastin-induced cell injury model. Scale bar: 200 μm. All results were shown as the mean ± SEM from three independent experiments, ns means the difference is not statistically significant, **p < 0.01, and ***p < 0.001.

Article Snippet: Erastin, lapatinib, Fer-1, liproxstatin-1 (Lip-1) and Ras-selective lethal small molecule 3 (RSL3) were obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Lapatinib protects against epileptic seizures via halting glutathione peroxidase 4-dependent ferroptosis

doi: 10.1101/2020.05.28.120733

Figure Lengend Snippet: Lapatinib inhibited neuronal ferroptosis by upregulating GPX4. (a) Western blot analysis of ferroptosis-related proteins including GPX4, SLC7A11, 5-LOX and ACSL4 pretreated with lapatinib (10 μM) and Fer-1 (12.5 μM) for 2 h in Glu- or erastin-induced HT22 cell injury model. (b) Immunofluorescence analysis of GPX4 expression after pretreatment for 2 h with lapatinib (10 μM), followed by exposure to Glu or erastin for another 8 h in HT22 cells. Scale bar: 50μm. (c) The level of GSH was assessed following lapatinib (10 μM) pretreatment for 2 h. (d, e) To observe the effect between lapatinib (10 μM) and RSL3 (1 nM) co-intervention group and lapatinib intervention alone group on the Glu- or erastin-induced cell injury model. Scale bar: 200 μm. All results were shown as the mean ± SEM from three independent experiments, ns means the difference is not statistically significant, **p < 0.01, and ***p < 0.001.

Article Snippet: Erastin, lapatinib, Fer-1, liproxstatin-1 (Lip-1) and Ras-selective lethal small molecule 3 (RSL3) were obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Western Blot, Immunofluorescence, Expressing

Journal: Journal of cellular physiology

Article Title: Blocking Fibrotic Signaling In Fibroblasts from Patients with Carpal Tunnel Syndrome

doi: 10.1002/jcp.25901

Figure Lengend Snippet: Gene expression analysis using qRT-PCR of known CTS fibrosis marker genes Col1, 3, CTGF, SERPINE1 upon cytokine receptor level inhibition. CTS fibroblasts were treated with TGF-β1 and TβRI (SD208), PDGFR (AG1296), EGFR (Lapatinib) and VEGFR (Axitinib) inhibitor for 24 hours. Gene expression is normalized to vehicle control. All data are expressed as mean ± SE. n=5. (* indicates p < 0.05, # indicates p < 0.01).

Article Snippet: Lapatinib (IC50: 10.8 nM) (Selleck Chemical, Houston, TX, S1028) is a small molecule kinase inhibitor that targets both EGFR1 and 2 (Her2) ( Moy et al., 2007 ).

Techniques: Gene Expression, Quantitative RT-PCR, Marker, Inhibition, Control

Journal: Journal of cellular physiology

Article Title: Blocking Fibrotic Signaling In Fibroblasts from Patients with Carpal Tunnel Syndrome

doi: 10.1002/jcp.25901

Figure Lengend Snippet: Smad reporter activity comparing cytokine receptor inhibition. Comparison of CTS fibroblasts treated with TGF-β1 and TβRI (SD208), PDGFR (AG1296), EGFR (Lapatinib) and VEGFR (Axitinib) inhibitor for 24 hours. Data are expressed as relative fold change from vehicle control. All data are expressed as mean ± SE. n=3. (# indicates p < 0.01).

Article Snippet: Lapatinib (IC50: 10.8 nM) (Selleck Chemical, Houston, TX, S1028) is a small molecule kinase inhibitor that targets both EGFR1 and 2 (Her2) ( Moy et al., 2007 ).

Techniques: Activity Assay, Inhibition, Comparison, Control

Journal: Oncotarget

Article Title: Staurosporine, an inhibitor of hormonally up-regulated neu-associated kinase

doi: 10.18632/oncotarget.26311

Figure Lengend Snippet: ( A ) IC 50 value was generated by evaluating viability of JIMT-1 cells treated with increasing doses of lapatinib to show these cells are inherently resistant to lapatinib. ( B ) IC 50 value was generated by evaluating viability of JIMT-1 cells treated with increasing doses of STU. ( C ) Dose response curves and IC 50 value were generated by evaluating viability of JIMT-1 cells treated with increasing doses of STU in combination with 100 nM lapatinib. ( D ) Combination index analysis indicates that STU is synergistic with 100 nM lapatinib at all concentrations evaluated, with a score below “1” indicating synergy. ( E ) JIMT-1 cells were treated with either DMSO or 25 nM STU for 24 hours. Immunoblotting for PKC activity was performed using anti-phospho-PKC, anti-phospho-PKC substrate, and anti-PKC antibodies. ( F ) To assess colony formation, JIMT-1 cells were initially plated in normal growth medium, followed by next day replacement of media containing drug (DMSO, 100 nM lapatinib, 25 nM STU, or combination 100 nM lapatinib plus 25 nM STU). Cells were treated with drugs for 3 days, followed by replacement with normal growth media for the next 21 days. At day 21, colonies were stained with crystal violet and quantified. ( G ) To assess mammosphere formation and renewal, JIMT-1 cells were plated in media containing either DMSO, 25 nM STU, 100 nM lapatinib, or combination 25 nM STU plus 100 nM lapatinib. Cells were maintained in drug for 14 days, then quantified and imaged. To assess renewal, primary mammospheres were trypsinized and re-plated in drug containing media at equal numbers. Mammosphere formation was quantified 14 days after primary mammospheres were plated for renewal. ( H ) JIMT-1 cells were injected into the mammary fat pad of immunocompromised mice to assess mammary tumor growth. Mice received placebo or 100 mg/kg lapatinib twice a week for the duration of the experiment. ( I ) To assess combined delivery of STU and lapatinib, JIMT-1 cells were injected into the mammary fat pad of immunocompromised mice to assess mammary tumor growth. Mice were randomized into either placebo, STU (3 mg/kg), lapatinib (50 mg/kg), or combination STU plus lapatinib treatment groups. When tumors reached a size of ∼65 mm 3 , treatments were initiated and administered twice weekly. Tumors were analyzed by student’s T -test at individual time points. * p < 0.001; ** p = 0.02.

Article Snippet: Lapatinib was purchased from Santa Cruz Biotech.

Techniques: Generated, Western Blot, Activity Assay, Staining, Injection

Journal: Cancers

Article Title: Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression

doi: 10.3390/cancers14051303

Figure Lengend Snippet: JAM-A antagonism reduces tumor size in a chick embryo xenograft model. ( A ) SUM-225 cells (2 × 10 6 ) were implanted onto the chorioallantoic membrane overlying a developing chick embryo and treated then and 4 days later with 15 µL PBS, 200 µM JBS2, and 0.1 µM lapatinib or JBS + lapatinib. ( A ) Upon xenograft harvesting, the number of grossly visible tumors was observed to be less in treated relative to control conditions according to the following hierarchy: PBS > lapatinib > JBS2 > JBS2 + lapatinib ( n = 5–8 eggs per condition). No embryonic death was observed for JBS2-treated eggs, while there were 1–3 embryonic deaths in the other conditions as follows: PBS < lapatinib < JBS2 + lapatinib (*** p < 0.0001). ( B ) Representative image of a gross xenograft tumor (arrowhead) before harvesting from the CAM. The boundary of the silicon ring is shown as a dashed line. ( C ) Representative images of formalin-fixed paraffin-embedded xenograft tumor sections (4 μm) stained with hematoxylin/eosin.

Article Snippet: These were treated for 28 days as follows: PBS ( n = 8; daily intra-peritoneal/i.p. injection at equivalent volume to JBS2, typically 120–150 μL), JBS2 ( n = 9; 10 mg/kg daily i.p), lapatinib tosylate (Carbosynth Ltd., Berkshire, UK; n = 8; 100 mg/kg oral gavage on 5 consecutive days per week), JBS2 plus lapatinib ( n = 8; 10 mg/kg and 100 mg/kg respectively).

Techniques: Membrane, Control, Formalin-fixed Paraffin-Embedded, Staining

Journal: Cancers

Article Title: Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression

doi: 10.3390/cancers14051303

Figure Lengend Snippet: JAM-A antagonism reduces tumor progression in an mfp tumor xenograft model. ( A ) SUM-225- luc tumor xenografts grown to a volume of 100 mm 3 in the mfps of NOD/SCID mice were treated for 4 weeks with PBS, JBS2, lapatinib, or JBS2 + lapatinib and volumetrically measured twice/week ( n = 6, 9, 6, 8 mice, respectively). JBS2, lapatinib, and JBS2+lapatinib all significantly reduced tumor growth over time versus PBS-treated tumors (*** p < 0.001). Co-administration of JBS2 with the positive control drug lapatinib significantly shortened the time until lapatinib reached its maximal effect (* p < 0.05, comparing volumes in lapatinib versus JBS2+lapatinib tumors at 1 week post treatment). ( B ) Tumor bioluminescence was imaged once-weekly by in vivo imaging system (IVIS), and mean values over the entire treatment period (weeks 1–4 inclusive) expressed as flux (pixels/second; p/s) were plotted. Graph shows maximum and minimum values with a line at the mean bioluminescence for each treatment group. JBS2 significantly reduced mean tumor bioluminescence (* p < 0.05; PBS versus JBS2 and JBS2 versus JBS2 + Lapatinib). ( C ) FFPE tumor sections were stained with hematoxylin and eosin (top panel) or immunohistochemically assessed for the expression of JAM-A (middle panel) or cytokeratin (CK)-5/6 (lower panel). ( D ) JAM-A expression (measured semi-quantitatively) was reduced in tumor sections from mice treated with JBS2 relative to PBS (* p < 0.05). ( E ) The percentage positivity for cytokeratin 5/6 expression in xenograft tumor cells was not altered between treatment groups ( n = 9–11 mice per group), where ST refers to sub-threshold tumors that never reached 100 mm 3 and were thus not randomized to receive treatment. All treatments reduced the percentage of CK5/6-positive cells in xenograft basement membranes (BM); ( F ) a surrogate for tumor cell invasion.

Article Snippet: These were treated for 28 days as follows: PBS ( n = 8; daily intra-peritoneal/i.p. injection at equivalent volume to JBS2, typically 120–150 μL), JBS2 ( n = 9; 10 mg/kg daily i.p), lapatinib tosylate (Carbosynth Ltd., Berkshire, UK; n = 8; 100 mg/kg oral gavage on 5 consecutive days per week), JBS2 plus lapatinib ( n = 8; 10 mg/kg and 100 mg/kg respectively).

Techniques: Positive Control, In Vivo Imaging, Staining, Expressing

Journal: Cancers

Article Title: Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression

doi: 10.3390/cancers14051303

Figure Lengend Snippet: JAM-A and HER2 antagonism evoke multiple proteomic changes in mfp tumor xenografts. Fresh-frozen tissues harvested from the SUM-225 mouse mfp model of breast cancer (treatment groups: PBS, JBS2, lapatinib, JBS2+lapatinib) were subjected to proximity ligation assay proteomic analysis using an oncology-centered 92-protein array panel. Seven individual protein targets changed significantly between conditions in the 92-protein array (CAIX, MUC-16, ESM-1, TFPI-2, VEGFA, IL-6, HGF; * p < 0.05).

Article Snippet: These were treated for 28 days as follows: PBS ( n = 8; daily intra-peritoneal/i.p. injection at equivalent volume to JBS2, typically 120–150 μL), JBS2 ( n = 9; 10 mg/kg daily i.p), lapatinib tosylate (Carbosynth Ltd., Berkshire, UK; n = 8; 100 mg/kg oral gavage on 5 consecutive days per week), JBS2 plus lapatinib ( n = 8; 10 mg/kg and 100 mg/kg respectively).

Techniques: Proximity Ligation Assay, Protein Array