Journal: Cancers

Article Title: Mutant p53 L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to Mortaparib Plus , a Multimodal Small Molecule Inhibitor

doi: 10.3390/cancers13123043

Figure Lengend Snippet: Mortaparib Plus is toxic to MCF-7, but not T47D, Luminal-A breast cancer cells. ( A ) MTT-cell viability assays for control and 48 h-Mortaparib Plus -treated cells showed a dose-dependent cytotoxicity in MCF-7 and T47D with a milder effect on the non-tumorigenic lung fibroblasts (TIG-3 and MRC-5). Chemical structure of Mortaparib Plus is shown. ( B ) CV-cell viability assay for control and 48 h-Mortaparib Plus -treated cells showed dose-dependent cytotoxicity in MCF-7 cells; T47D showed poor response and were similar to MRC-5 and TIG-3. ( C ) Phase-contrast micrographs of control and 48 h-Mortaparib Plus -treated MCF-7 cells showed a stressed phenotype (condensation and blebbing morphologies); T47D, MRC-5 and TIG-3 cells did not show such stress/cytotoxic morphology ( C , D ). The quantified cell viability data represents mean ± SD obtained from independent biological replicates; p -values were calculated using unpaired Student’s t -test. * ≤0.05, ** ≤0.01, *** ≤0.001 and **** ≤0.0001 represent significant, very significant, highly significant and extremely significant, respectively.

Article Snippet: Luminal-A breast cancer cell line (T47D) was purchased from the DS Pharma Biomedical Co. Ltd., Japan.

Techniques: Control, Viability Assay

Journal: Cancers

Article Title: Mutant p53 L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to Mortaparib Plus , a Multimodal Small Molecule Inhibitor

doi: 10.3390/cancers13123043

Figure Lengend Snippet: Mortaparib Plus abrogated the interaction of mortalin with p53 in vitro causing the reactivation of the wild-type p53 signaling. Co-immunoprecipitation (Co-IP) analyses using a rabbit monoclonal anti-mortalin antibody ( A , B ) showed a decrease in p53 fractions (wild type and mutant) in mortalin complexes immunoprecipitated from Mortaparib Plus -treated cells. Full uncropped blots for the Co-IP experiments are shown in . Control and 48 h-Mortaparib Plus -treated cells were analyzed for the expression levels of p53, mortalin and several p53 downstream target genes by Western blotting ( C ). An accumulation in p53 levels with the overexpression of its downstream target genes (p21 WAF1 , PUMA and BAX) was observed in 48 h-Mortaparib Plus -treated MCF-7, but not T47D, cells as compared to the untreated control. Control and Mortaparib Plus -treated normal lung fibroblasts were analyzed for the expression levels of p53 and p21 by Western blotting ( D ). TIG-3 and MRC-5 normal lung fibroblasts also did not show a significant increase in p53 or its downstream effector p21 WAF1 . Full uncropped blots for the Western blotting experiments are shown in . The quantified data represent mean ± SD obtained from independent biological replicates; p -values were calculated using unpaired Student’s t -test. ns > 0.05, * ≤0.05, ** ≤0.01 and *** ≤0.001 represent non-sgnificant, significant, very significant and highly significant, respectively.

Article Snippet: Luminal-A breast cancer cell line (T47D) was purchased from the DS Pharma Biomedical Co. Ltd., Japan.

Techniques: In Vitro, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Control, Expressing, Western Blot, Over Expression

Journal: Cancers

Article Title: Mutant p53 L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to Mortaparib Plus , a Multimodal Small Molecule Inhibitor

doi: 10.3390/cancers13123043

Figure Lengend Snippet: Mortaparib Plus caused nuclear translocation of p53. The sub-cellular localization of p53 and mortalin in the control and 48 h-Mortaparib Plus -treated cells was examined by immunocytochemistry ( A , B ). Mortaparib Plus -treated MCF-7 cells showed p53 enrichment in the nuclei as compared to the nuclei of untreated control. T47D cells showed the mutant p53 L194F in the nuclei of both the 48 h-Mortaparib Plus -treated and untreated ones. The shift of the mortalin staining pattern from perinuclear in control to pan-cytoplasmic in Mortaparib Plus -treated MCF-7 and T47D cells was also observed (shown by white arrows). The transcriptional activation function of p53 was examined by the p53 wild-type specific luciferase reporter (PG13-Luc) assay ( C ). The wild-type p53-dependent luciferase reporter activity showed a dramatic increase in Mortaparib Plus -treated MCF-7, but not T47D, cells. Scale bar: 50 μm. The quantified data represent mean ± SD obtained from independent biological replicates; p -values were calculated using unpaired Student’s t -test. ns > 0.05, * ≤0.05, ** ≤0.01, and **** ≤0.0001 represent significant, non-significant, very significant, and extremely significant, respectively.

Article Snippet: Luminal-A breast cancer cell line (T47D) was purchased from the DS Pharma Biomedical Co. Ltd., Japan.

Techniques: Translocation Assay, Control, Immunocytochemistry, Mutagenesis, Staining, Activation Assay, Luciferase, Activity Assay

Journal: Cancers

Article Title: Mutant p53 L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to Mortaparib Plus , a Multimodal Small Molecule Inhibitor

doi: 10.3390/cancers13123043

Figure Lengend Snippet: Mortaparib Plus induced apoptosis and G 2 /M cell cycle arrest only in the wild type p53- harboring Luminal-A breast cancer cells. Flow cytometric analyses for control and 48 h Mortaparib Plus -treated cells showed an increase in the early apoptotic cell populations in Mortaparib Plus -treated MCF-7, but not T47D, cells ( A ). Using the same flow cytometric platform, cell cycle progression analyses for control and 48 h-Mortaparib Plus -treated cells revealed that Mortaparib Plus (3 μM) induced a strong G 2 /M cell cycle arrest in MCF-7 cells; Mortaparib Plus -treated T47D cells were not arrested at either G 1 /S or G 2 /M cell cycle phases ( B ). The quantified data represent mean ± SD obtained from independent biological replicates; p -values were calculated using unpaired Student’s t -test. ns > 0.05, * ≤0.05, ** ≤0.01, and *** ≤0.001 represent non-significant, significant, very significant, and highly significant, respectively.

Article Snippet: Luminal-A breast cancer cell line (T47D) was purchased from the DS Pharma Biomedical Co. Ltd., Japan.

Techniques: Control

Journal: Cancers

Article Title: Mutant p53 L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to Mortaparib Plus , a Multimodal Small Molecule Inhibitor

doi: 10.3390/cancers13123043

Figure Lengend Snippet: Mortaparib Plus caused an accumulation of PAR polymer and DNA damage in T47D (p53 L194F ) cells. The levels of the full-length PARP1, the cleaved 89-kDa fragment of PARP1 and PAR polymer were analyzed in control and 48 h Mortaparib Plus -treated cells by Western blotting ( A ). The levels of the full-length PARP1 protein, PAR polymer and the phosphorylated H2A histone variant X (γH2AX) protein were measured by immunocytochemistry ( B – D ). No significant change in the levels of the full-length PARP1 was observed in Mortaparib Plus -treated T47D cells as compared to the untreated control. A significant accumulation of PAR polymer and the foci of the phosphorylated H2A histone variant X (γH2AX) were observed in the Mortaparib Plus -treated T47D cells as compared to the untreated control. Total cellular ATP concentrations in DMSO control and Mortaparib Plus -treated T47D cells were measured using a luminescent ATP detection method ( E ). ATP levels were decreased in Mortaparib Plus -treated T47D cells compared to the DMSO control-treated cells. Full uncropped blots for the Western blotting experiments are shown in . Scale bar: 50 μm. The quantified data represent mean ± SD obtained from independent biological replicates; p -values were calculated using unpaired Student’s t -test. ns > 0.05, * <0.05, ** <0.01 and *** <0.001 represent non-significant, significant, very significant, and highly significant, respectively.

Article Snippet: Luminal-A breast cancer cell line (T47D) was purchased from the DS Pharma Biomedical Co. Ltd., Japan.

Techniques: Polymer, Control, Western Blot, Variant Assay, Immunocytochemistry

Journal: Cancers

Article Title: Mutant p53 L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to Mortaparib Plus , a Multimodal Small Molecule Inhibitor

doi: 10.3390/cancers13123043

Figure Lengend Snippet: Mortaparib Plus treated cells did not show translocation of apoptosis-inducing factor (AIF) to the nuclei. AIF’s expression level in control and 48 h Mortaparib Plus -treated T47D cells was examined by Western blotting ( A ). There were no significant changes in AIF’s expression levels in control and Mortaparib Plus -treated T47D cells. AIF’s subcellular localization in control and 48 h-Mortaparib Plus -treated T47D and MCF-7 cells was analyzed by immunocytochemistry. Co-localization of mortalin and AIF was observed in T47D cells ( B ). MCF-7 cells showed a low level of expression of AIF as compared to T47D cells; its colocalization with mortalin was observed and more prominent in Mortaparib Plus -treated cells ( C ). Full uncropped blots for the Western blotting experiments are shown in . Scale bar: 50 mm. The quantified data represent mean ± SD obtained from independent biological replicates; p -values were calculated using unpaired Student’s t -test. ns > 0.05 represents non-significant.

Article Snippet: Luminal-A breast cancer cell line (T47D) was purchased from the DS Pharma Biomedical Co. Ltd., Japan.

Techniques: Translocation Assay, Expressing, Control, Western Blot, Immunocytochemistry

Journal: Cancers

Article Title: Mutant p53 L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to Mortaparib Plus , a Multimodal Small Molecule Inhibitor

doi: 10.3390/cancers13123043

Figure Lengend Snippet: Mortaparib Plus treatment did not abrogate AIF-mortalin interaction in T47D (p53 L194F ). AIF–mortalin interaction in both control and 48 h Mortaparib Plus -treated T47D cells were analyzed by co-immunoprecipitation ( A ). An increase in AIF fractions was observed in the mortalin complexes immunoprecipitated from Mortaparib Plus -treated T47D cells as compared to both the untreated and IgG controls. Mortalin knockdown could not relocate AIF to the nuclei of T47D cells (p53 L194F ). Mortalin and AIF subcellular localizations were examined by immunocytochemistry after mortalin knockdown using a mortalin-specific shRNA (shRNA2166) ( B ). Mortalin knockdown was not sufficient to liberate AIF from mortalin complex even after the Mortaparib Plus -induced PAR accumulation. NT represents Non-Transfected cells. Full uncropped blots for the Co-IP experiments are shown in . Scale bar: 50 mm. The quantified data represent mean ± SD obtained from independent biological replicates; p -values were calculated using unpaired Student’s t -test. ns > 0.05 and ** ≤0.01 represents non-significant and very significant.

Article Snippet: Luminal-A breast cancer cell line (T47D) was purchased from the DS Pharma Biomedical Co. Ltd., Japan.

Techniques: Control, Immunoprecipitation, Knockdown, Immunocytochemistry, shRNA, Transfection, Co-Immunoprecipitation Assay