Journal: Oncology reports

Article Title: Targeting P16INK4A in uterine serous carcinoma through inhibition of histone demethylation.

doi: 10.3892/or.2019.7067

Figure Lengend Snippet: Figure 1. Differential expression of P16INK4A in tumor samples. (A) Representative images of P16INK4A positivity in USC, P16INK4A negativity in UEC and P16INK4A negativity in normal endometrium. The normal tissue was obtained from 8 patients with leiomyoma who receiving hysterectomy procedures. Scale bar, 50 µm. (B) Percentage of P16INK4A‑positive cases among 33 USC and 88 UEC samples. **P<0.01 (χ2 test). (C) Western blot analysis of 6 endometrial cell lines (AN3CA, Hec1A, Hec108, Nou‑1, EFE‑184 and ETN‑1). Vinculin was used as a loading control. (D) The reverse transcription‑quantitative polymerase chain reaction results of the 6 endometrial cell lines (AN3CA, Hec1A, Hec108, Nou‑1, EFE‑184 and ETN‑1) were analyzed and shown as relative P16INK4A mRNA level of the Hec1A values, which were then converted as fold change. *P<0.05 and **P<0.01 [analysis of variance and a post hoc test (Student‑Newman‑Keuls)]. (E) Response of different endometrial cell lines to the CDK4/6 inhibitor palbociclib. The viability of ETN‑1, EFE‑184, Hec‑1A, AN3CA, Hec108 and Nou‑1 cells was measured with a Cell Counting Kit‑8 assay following treatment with vehicle (dimethyl sulfoxide) and palbociclib (0.5, 1, 2.5, 5 and 10 µM) for 72 h. The IC50 are depicted. P16INK4A, cyclin-dependent kinase inhibitor 2A; CDK4, cyclin-dependent kinase 4; UEC, uterine endometroid carcinoma; USC, uterine serous carcinoma; H3K27, histone 3 lysine 27; KDM6B, histone lysine demethylase 6B; IC50, half‑maximal inhibitory concentration; RB, retinoblastoma.

Article Snippet: Palbociclib was purchased from Selleck Chemicals (Houston, TX, USA; cat. no. S1579), and GSK-J4 was obtained from MedChemExpress (Shanghai, China; cat. no. HY-15648B).

Techniques: Quantitative Proteomics, Western Blot, Control, Polymerase Chain Reaction, CCK-8 Assay, Concentration Assay

Journal: Oncogene

Article Title: RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models

doi: 10.1038/s41388-022-02362-2

Figure Lengend Snippet: A Heat map representing the relative growth rate of MCF7 and T47D cells in the presence of drug library at two different concentrations (100 nM and 500 nM) from cluster 1. B Western blot analysis on the indicated proteins from MCF7 and T47D cells that were treated with everolimus (250 nM) and alpelisib (250 nM) up to 48 H. C Western blot analysis to investigate the effect of alpelisib (250 nM) on cell cycle proteins from T47D and T47D-D1/K4 that were treated up to 48 H. D Live cell imaging to monitor the growth of MCF7-WT, MCF7-D1/K4, T47D-WT, and T47D-D1/K4 cells treated with different pairwise combination of palbociclib (50 nM), fulvestrant (100 nM), everolimus (125 nM) and alpelisib (125 nM). Error bars represent mean and SD from triplicates. Experiments were done at two independent times. E Western blot on the indicated proteins from MCF7-WT, MCF7-D1/K4, T47D-WT and T47D-D1/K4 that were treated with palbociclib (50 nM) + fuvlestrant (100 nM) and alpelisib (125 nM) + fulvestrant (100 nM) for 48 h. F Growth curves illustrating the effect of indicated combination treatments involving palbociclib (50 nM), fulvestrant (100 nM), everolimus (125 nM) and alpelisib (125 nM) in MCF7-RB-del and T47D-RB-del cells. Mean and SD were calculated from triplicates. G Western blot analysis to demonstrate the differential effect of palbociclib (50 nM) + Everolimus (125 nM) and palbociclib (50 nM)+ alpelisib (125 nM) on cell cycle proteins. H Heat map to demonstrate the synergistic effect between the combination of palbociclib with everolimus (Palb/Evero) and alpelisib (Palb/Alpe) on BrdU incorporation at the indicated doses in MCF-WT, MCF7-RB-del, T47D-WT, and T47D-RB-del cells. The values in heat map represent the mean from triplicates. BrdU incorporation was determined in CAMA-1 WT and CAMA-1-RB-del cells treated with the palbociclib (palbo) in combination with everolimus (evero) and fulvestrant (fulve) at indicated concentrations for 72 h. Error bars represent SD from triplicates (*p < 0.05, **p < 0.01 as determined by two-way ANOVA). I PFS of patients receiving CDK4/6i in combination with endocrine therapy with tumors harboring WT RB (n = 68) and RB-deletion mutation (n = 3). All p values were indicated.

Article Snippet: Palbociclib, Alisertib, and Birinapant were purchased from MedChemExpress (NJ, USA).

Techniques: Drug discovery, Western Blot, Live Cell Imaging, BrdU Incorporation Assay, Mutagenesis

Journal: Oncogene

Article Title: RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models

doi: 10.1038/s41388-022-02362-2

Figure Lengend Snippet: A Heat maps representing the relative cell viability of MCF7-WT, MCF7-RB-del, T47D-WT and T47D-RB-del cells in the presence of drug library (100 nM) following the pretreatment with birinapant (250 nM) from clusters 1 & 2. B Cell viability assays from MCF7 and T47D WT/RB-del that were treated with birinapant in combination with the indicated drugs at the indicated concentrations up to 6 days. Mean and SD were calculated from triplicates. Experiment was done at 2 independent times. (*p < 0.05, **p < 0.01 as determined by two-way ANOVA). C Crystal violet staining from MCF7-WT, MCF7-RB-del, T47D-WT and T47D-RB-del cells that were treated with Alpelisib (100 nM) in combination with DMSO or birinapant (500 nM). Cells were allowed to form colonies up to 15 days. D Live cell imaging to monitor the proliferation of MCF7-WT and RB-del cells that were treated with DMSO, birinapant (500 nM) and palbociclib (50 nM) + fulvestrant (100 nM) ± birinapant. The live cell imaging was terminated, and the cells were subjected to CTG assay to determine the viable cells. Mean and SD were calculated from 6 replicates. (**p < 0.01, ***p < 0.001 as determined by two-way ANOVA).

Article Snippet: Palbociclib, Alisertib, and Birinapant were purchased from MedChemExpress (NJ, USA).

Techniques: Drug discovery, Staining, Live Cell Imaging, CTG Assay

Journal: NPJ Precision Oncology

Article Title: IL-6 predicts CDK4/6 inhibitor resistance, identifying STAT3 as a target in HR + /HER2-negative metastatic breast cancer

doi: 10.1038/s41698-025-01041-1

Figure Lengend Snippet: A Relative gene expression of IL-6 by quantitative (q)RT-PCR analysis in MCF7 parental (Par) and resistant (Res) cell lines after shRNA knockdown of IL-6 by one of two clones (shIL-6 #1 and shIL-6 #2) or control scrambled shRNA (shSCR). Cells were grown in culture for 3 days. B Relative gene expression of STAT3 by qRT-PCR analysis. C Secreted IL-6 levels measured by ELISA and normalized based on the number of cells (relative concentration [conc]). D Western blot analysis of pSTAT3 (Y705) and total STAT3 after 3 days in culture. E Dose-response curves of treatment with 0.01–12 μM palbociclib for 6 days followed by 6 days of recovery compared with MCF7 Par shSCR. Number of cells/growth was assessed using the crystal violet assay. Data were normalized to DMSO (100%), plotted, and analyzed using nonlinear regression on GraphPad Prism 9. Dashed line and bar graphs depict half-maximal inhibitory concentration (IC 50 ) values. F Dose-response curves after 24 h of estrogen deprivation followed by re-addition of 10 nM beta-estradiol and treatment with varying concentrations (0.01–12 μM) of fulvestrant for 2 days compared with MCF7 Par shSCR. Number of cells/growth was assessed using the crystal violet assay as in panel E. G Dose-response curves with 0.01–12 μM TTI-101 for 3 days followed by 9 days of recovery. Number of cells/growth was assessed using the crystal violet assay as in panel ( E ). H qRT-PCR analysis of estrogen responsive genes—ER and PgR. I qRT-PCR analysis of epithelial-mesenchymal transition (EMT) markers N-cadherin and Vimentin. J qRT-PCR analysis of transcription factors related to breast cancer stem cell-like markers CD44 and ALDH1. K Western blot analysis of estrogen receptor (ERa), PgR, EMT markers, and G1/S transition proteins. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Palbociclib isethionate (MedChemExpress) was diluted in 0.5 W/V% methyl cellulose 400 (FUJIFILM Wako Chemicals), TTI-101 was diluted in 60:40 Labrasol:PEG400 with brief sonication for about 2 min, and vehicle was a 50:50 mixture of 0.5 W/V% methyl cellulose 400 and 60:40 Labrasol:PEG400.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, shRNA, Knockdown, Clone Assay, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Crystal Violet Assay

Journal: NPJ Precision Oncology

Article Title: IL-6 predicts CDK4/6 inhibitor resistance, identifying STAT3 as a target in HR + /HER2-negative metastatic breast cancer

doi: 10.1038/s41698-025-01041-1

Figure Lengend Snippet: A , B Dose-response curves in MCF7 and T47D parental (Par) control scrambled shRNA (shSCR), shIL-6 #1, and shIL-6 #2 cells, depicting the effect of treatment with 0.01–12 μM palbociclib for 6 days followed by 6 days of recovery ( A ) and after 24 h of estrogen deprivation followed by re-addition of 10 nM beta-estradiol and treatment with varying concentrations (0.01–12 μM) of fulvestrant for 2 days ( B ). Dashed line depicts half-maximal inhibitor concentration (IC 50 ) values. C , D Bar graphs depicting the average IC 50 values in MCF7 and T47D parental shSCR, shIL-6 #1, and shIL-6 #2 cells treated with palbociclib ( C ) and fulvestrant ( D ) as indicated in A , B. E , F Doubling time every 3 days for 21 days showing the effect of shRNA knockdown of IL-6 on the proliferation of MCF7 and T47D parental cell lines. G , H MCF7 parental ( G ) and T47D ( H ) shSCR, shIL-6 #1, and shIL-6 #2 cells were treated with 1 μM palbociclib every 3 days for 21 days with measurements taken every 3 days as indicated in G for cell growth and IL-6 secreted protein levels (ELISA). I Representative images of senescence-associated ß-galactosidase (SA-βgal) staining in MCF7 parental shSCR, shIL-6 #1, and shIL-6 #2 cells treated with or without 1 μM palbociclib every 3 days for 21 days. Quantification of SA-βgal+ cells was calculated every 3 days relative to number of cells. J Representative images of SA-βgal staining in MCF7 resistant (Res) and T47D Res shSCR, shIL-6 #1, and shIL-6 #2 cells treated with or without 1 μM palbociclib every 3 days for 6 days. Quantification of SA-βgal+ cells was calculated at day 6 relative to number of cells. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Palbociclib isethionate (MedChemExpress) was diluted in 0.5 W/V% methyl cellulose 400 (FUJIFILM Wako Chemicals), TTI-101 was diluted in 60:40 Labrasol:PEG400 with brief sonication for about 2 min, and vehicle was a 50:50 mixture of 0.5 W/V% methyl cellulose 400 and 60:40 Labrasol:PEG400.

Techniques: Control, shRNA, Concentration Assay, Knockdown, Enzyme-linked Immunosorbent Assay, Staining

Journal: NPJ Precision Oncology

Article Title: IL-6 predicts CDK4/6 inhibitor resistance, identifying STAT3 as a target in HR + /HER2-negative metastatic breast cancer

doi: 10.1038/s41698-025-01041-1

Figure Lengend Snippet: A , B Fold change in the tumor volume in female nude mice bearing either PR-1 ( A ) or PR-4 ( B ) after treatment with combination of palbociclib (palbo) with TTI-101, and vehicle up to 74 days (PR-1) and 52 days (PR-4). The Tukey multiple comparisons test determined p values. C , D Relative change in tumor volume at the treatment endpoint for PR-1 ( C ) and PR-4 ( D ) for each individual mouse. E , F Event-free survival analysis of PR-1 ( E ) and PR-4 ( F ) PDX models treated as described in A-B, calculated based on the days on treatment when fold change in tumor volume reached 1.5 (PR-1) or 7 (PR-4). The Mantel-Cox log-rank test determined p values. G , H Quantification (left panel) and representative images (right panel) of Ki67 immunohistochemical staining at the treatment endpoint. The Tukey multiple comparisons test determined p values. I , J White blood cell count (WBC), red blood cell count (RBC), and neutrophils measured from blood collected at the treatment endpoint. Gray area indicates normal ranges for non–tumor-bearing female nude mice. K , L Change in body weight at the indicated days over the course of treatment. M , N Level of TTI-101 accumulation (ng/g) evaluated by LC-MS/MS in tumor lysates collected 4 h after the last drug was administered at the treatment endpoint in PR-1 and PR-4. ND=not detected. O . Quantification (left panel) and representative images (right panel) of pSTAT3 (Y705) immunohistochemical staining at treatment end point with the indicated treatments. Tukey’s multiple comparisons test determined p -values. * p < 0.05. P , Q Luminex analysis of tumor lysates for pSTAT3 and STAT3 at treatment end point with the indicated treatments in PR-1 (P) and PR-4 (Q). MFI = Mean Fluorescent Intensity, * p < 0.05, tumor lysates were normalized to 1ug/uL. R Tumor human IL-21 (hIL-21) levels as measured by the ProcartaPlex Human Immune Monitoring 65-plex Luminex analysis of PR-1 and PR-4 PDX tumor lysate samples collected at the treatment endpoint. S Hierarchical clustering heat map with the z-scores of significant differentially expressed proteins with adjusted p < 0.3 between combination (Palbo+TTI-101) and vehicle from reverse-phase protein array analysis of PR-1 PDX tumor lysates collected at the treatment endpoint. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Palbociclib isethionate (MedChemExpress) was diluted in 0.5 W/V% methyl cellulose 400 (FUJIFILM Wako Chemicals), TTI-101 was diluted in 60:40 Labrasol:PEG400 with brief sonication for about 2 min, and vehicle was a 50:50 mixture of 0.5 W/V% methyl cellulose 400 and 60:40 Labrasol:PEG400.

Techniques: Immunohistochemical staining, Staining, Cell Characterization, Liquid Chromatography with Mass Spectroscopy, Luminex, Protein Array

Journal: International Journal of Molecular Sciences

Article Title: Differential Expression of PD-L1 during Cell Cycle Progression of Head and Neck Squamous Cell Carcinoma

doi: 10.3390/ijms222313087

Figure Lengend Snippet: Efficacy of cell cycle inhibitors. ( A ) Inhibition of cell proliferation due to the incubation with cell cycle inhibitors. An MTT viability assay was used for IC 50 determination. Here, PCI 1 was chosen as the representative cell line with nocodazole (G2/M phase) treatment. Photo documentation after four days of inhibitor treatment confirmed the inhibition of cell proliferation. PCI 13 was chosen as the representative cell line with aphidicolin (S phase) treatment. Images are shown at four- and 20-fold magnification. ( B ) Cell cycle analysis via flow cytometry after inhibitor treatment. Flow cytometry confirmed that palbociclib treatment resulted in G1 phase, aphidicolin in S phase, and nocodazole in G2/M phase arrest. Here, PCI 13 was also chosen as the representative cell line. Incubation with DMSO instead of a specific inhibitor were used as controls. To quantify the DNA content, cells were incubated with DAPI. For analysis 5 × 10 4 cells were used per sample. The proportion of cells in a particular cell cycle phase is expressed as a percentage (%). ( C ) Effective concentrations of cell cycle inhibitors. Concentrations for palbociclib, aphidicolin, and nocodazole are listed in nanomolar (nM). High concentrations are highlighted in dark, whereas low concentrations are highlighted in light colors. Cell lines were subdivided into their epithelial and mesenchymal differentiation status and ranked according to their PD-L1 basal expression.

Article Snippet: Palbociclib (PD 0332991 isethionate), a potent cyclin-dependent CDK4 and CDK6 inhibitor that induces G1 cell cycle arrest, was obtained from Tocris Bioscience (Bristol, UK).

Techniques: Inhibition, Incubation, MTT Viability Assay, Cell Cycle Assay, Flow Cytometry, Expressing

Journal: Cell death & disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors.

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: Fig. 7 A hypothesis model depicted that PKA phosphorylated UBE3A to prevent UBE3A degrading PBRM1. DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.

Article Snippet: The CDK4/6 inhibitors (Palbociclib, HY-50767S, MedChemExpress, China, 200mg per kg bodyweight, oral administration) and RBPJ Inhibitor-1 (RIN1, HY-137471, MedChemExpress, China, 40 mg per kg bodyweight, intraperitoneal injection) were used for mice study.

Techniques: Expressing

Journal: Molecular cell

Article Title: Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein’s C-Terminal Helix

doi: 10.1016/j.molcel.2019.03.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Palbociclib , Santa Cruz Biotechnology , Cat#sc-478943.

Techniques: Purification, Produced, Virus, Recombinant, Selection, Membrane, Flow Cytometry, Expressing, Plasmid Preparation, Generated

Journal: Oncotarget

Article Title: Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq

doi: 10.18632/oncotarget.13666

Figure Lengend Snippet: ( A ) Gene expression levels of 4 cyclin-dependent kinases (CDK1, CDK2, CDK4 and CDK6) are analyzed for each of the 3 identified main groups. Data are given as standard box plots. CDK2 and CDK4 are consistently highly expressed in all groups. Positions of each CDK gene are shown on the mean SOM expression landscape indicated by arrows. ( B ) Growth inhibition curves are shown for treatments of a patient-derived melanoma short-term culture using inhibitors directed against CDK2 (purvanolol), CDK4 palbociclib, activated BRAF (vemurafenib), ERK1/2 (SCH772984), and MEK1/2 (cobimetinib).

Article Snippet: The following small molecule inhibitors were used: CDK4 inhibitor palbociclib (PD-0332991, SEL-S1116-5MG, Biozol, Eching, Germany), CDK2 inhibitor purvanolol (SEL-S7793-5MG, Biozol), BRAF inhibitor vemurafenib (PLX4032, SEL-S1267-10MG, Biozol), MEK1/2 inhibitor cobimetinib (RG7420, SEL-S8041-5MG, Biozol), and ERK1/2 inhibitor SCH772984 (SEL-S7101-5MG, Biozol).

Techniques: Gene Expression, Expressing, Inhibition, Derivative Assay