Journal: International Journal of Molecular Sciences

Article Title: A Novel CDK4/6 and PARP Dual Inhibitor ZC-22 Effectively Suppresses Tumor Growth and Improves the Response to Cisplatin Treatment in Breast and Ovarian Cancer

doi: 10.3390/ijms23052892

Figure Lengend Snippet: ZC-22 suppresses proliferation of breast and ovarian cancer cells. ( A ) IC 50 of ZC-22, Olaparib, and LY2835219 (Abemaciclib) in breast and ovarian cancer cells. Cells were treated with increasing concentrations of ZC-22, Olaparib, or LY2835219 for 6 days with replenishment of medium every 2 days. Cell proliferation was analyzed by CCK-8 assays. ( B ) Cell proliferation assay of breast and ovarian cancer cells with indicated treatment. MDA-MB-231 cells were treated with 2 μM Olaparib (OLA) or LY2835219 (LY-2) alone or together (OLA + LY-2) or with ZC-22 alone for 48 or 72 h. SUM-159, OVCAR5, and SKOV3 cells were treated with 0.5 μM OLA or LY-2 alone or together or with ZC-22 alone. Cell proliferation assay was analyzed by CCK-8 assays. Values are means ± SD, n = 3. ns, not significant, * p < 0.05, ** p < 0.01 and *** p < 0.001 for comparisons with the DMSO-treated group using an unpaired t -test.

Article Snippet: CDK4/6i Abemaciclib (LY2835219) was purchased from Eli Lily and Company (Indianapolis, IN, USA), PARPi Olaparib was from Astrazeneca (Wilmington, DE, USA), and cisplatin was from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: CCK-8 Assay, Proliferation Assay

Journal: International Journal of Molecular Sciences

Article Title: A Novel CDK4/6 and PARP Dual Inhibitor ZC-22 Effectively Suppresses Tumor Growth and Improves the Response to Cisplatin Treatment in Breast and Ovarian Cancer

doi: 10.3390/ijms23052892

Figure Lengend Snippet: ZC-22 inhibits CDK4/6 and PARP to induce cell-cycle arrest, DNA damage, and apoptosis in breast and ovarian cancer cells. ( A , B ) MDA-MB-231 cells were treated with 2 μM LY2835219 (LY-2) or ZC-22. OVCAR5 cells were treated with 0.5 μM LY-2 or ZC-22, for 24 h. The levels of CDK4/6 and p-Rb/Rb were determined by Western blot ( A ). Cell-cycle distribution was analyzed by flow cytometry ( B ). Values are means ± SD, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 for comparisons with the DMSO-treated group at each phase in an unpaired t -test. ( C ) Western blot analysis of PARP, PAR, γH2A.X, and H2A.X in MDA-MB-231 cells treated with 2 μM Olaparib (OLA) or ZC-22 for 48 h (Left), or OVCAR5 cells treated with 0.5 μM OLA or ZC-22 for 48 h (Right). ( D , E ) Representative pictures (left) and quantification of percentage (right) of γH2A.X-positive cells in MDA-MB-231 cells treated with 2 μM OLA or ZC-22 for 24 h ( D ), or OVCAR5 cells treated with 0.5 μM OLA or ZC-22 for 24 h ( E ). Values are means ± SD, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 for comparisons with the DMSO-treated group. p value was calculated using an unpaired t -test. ( F , G ) Representative pictures ( F ) and quantification of percentage ( G ) of apoptotic cells in MDA-MB-231 cells treated with 2 μM LY-2, OLA, or ZC-22 for 48 h or in OVCAR5 cells treated with 0.5 μM LY-2, OLA or ZC-22 for 48 h. Values are means ± SD, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the DMSO-treated group. p value was calculated using an unpaired t -test.

Article Snippet: CDK4/6i Abemaciclib (LY2835219) was purchased from Eli Lily and Company (Indianapolis, IN, USA), PARPi Olaparib was from Astrazeneca (Wilmington, DE, USA), and cisplatin was from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: A Novel CDK4/6 and PARP Dual Inhibitor ZC-22 Effectively Suppresses Tumor Growth and Improves the Response to Cisplatin Treatment in Breast and Ovarian Cancer

doi: 10.3390/ijms23052892

Figure Lengend Snippet: ZC-22 suppresses growth of breast cancer xenografts. Female NOD-SCID mice were injected subcutaneously with 2 × 10 6 of MDA-MB-231 cells and treated ip daily with 50 mg/kg of Olaparib (OLA) or LY2835219 (LY-2) alone or together (OLA + LY-2) or with ZC-22 alone starting on day 10 after injection of cells when tumors were 50–80 mm 3 in size. Tumor volumes were measured every 2–3 days. ( A ) Growth curve of the MDA-MB-231 xenograft tumors with indicated treatment. Values are means ± SEM, n = 6. ( B , C ) Pictures of ( B ) and quantification of the size of ( C ) the MDA-MB-231 xenograft tumors at the end points. Values are means ± SD, n = 6. ( D ) Immunohistochemical staining of Ki67 (top panels) and γH2A.X (bottom panels) in the MDA-MB-231 xenograft tumor tissues. ( E ) Quantification of Ki67 (Left) and γH2A.X (Right)-positive cells in immunohistochemical staining of the xenograft tumor tissues. Values are means ± SEM, n = 6. * p < 0.05, ** p < 0.01, and *** p < 0.001 in an unpaired t -test.

Article Snippet: CDK4/6i Abemaciclib (LY2835219) was purchased from Eli Lily and Company (Indianapolis, IN, USA), PARPi Olaparib was from Astrazeneca (Wilmington, DE, USA), and cisplatin was from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Injection, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: A Novel CDK4/6 and PARP Dual Inhibitor ZC-22 Effectively Suppresses Tumor Growth and Improves the Response to Cisplatin Treatment in Breast and Ovarian Cancer

doi: 10.3390/ijms23052892

Figure Lengend Snippet: ZC-22 increases the sensitivity of breast and ovarian cancer cells to cisplatin. MDA-MB-231 or OVCAR5 cells were treated with 2.5 μM cisplatin (DDP) alone or together with 2 μM Olaparib (OLA), LY2835219 (LY-2), or ZC-22 for the indicated time. ( A ) CCK-8 assays of MDA-MB-231 (left) and OVCAR5 (right) cells treated with DDP alone or together with OLA, LY-2, or ZC-22 for 48 or 72 h, n = 4. ( B ) EdU incorporation analysis of MDA-MB-231 (Up) and OVCAR5 (Down) cells treated with DDP alone or together with OLA, LY-2, or ZC-22 for 24 h. Representative pictures of EdU staining are shown in the left panels, and quantification of percentage of EdU-positive cells was presented in the bar graphs on the right, n = 3. ( C ) Western blot analysis of PARP, PAR, and γH2A.X/H2A.X in MDA-MB-231 (left) and OVCAR5 (right) cells treated with DDP alone or together with OLA or ZC-22 for 48 h. ( D ) Representative pictures (left) and quantification of percentage (right) of γH2A.X-positive cells in MDA-MB-231 (top panels) and OVCAR5 (bottom panels) cells treated with DDP alone or together with OLA or ZC-22 for 24 h, n = 3. ( E ) Western blot analysis of caspase 3 (cas3) cleavage in MDA-MB-231 (top) and OVCAR5 (bottom) cells treated with DDP alone or together with LY-2, OLA, or ZC-22 for 48 h. ( F , G ) Representative pictures ( F ) and quantification of percentage ( G ) of apoptotic cells in MDA-MB-231 (top panels) and OVCAR5 (bottom panels) cells treated with DDP alone or together with LY-2, OLA, or ZC-22 for 48 h, n = 3. Values in all panels are means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 in an unpaired t -test.

Article Snippet: CDK4/6i Abemaciclib (LY2835219) was purchased from Eli Lily and Company (Indianapolis, IN, USA), PARPi Olaparib was from Astrazeneca (Wilmington, DE, USA), and cisplatin was from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: CCK-8 Assay, Staining, Western Blot

Journal: Molecular Oncology

Article Title: CDK4/6 inhibitors induce breast cancer senescence with enhanced anti‐tumor immunogenic properties compared with DNA‐damaging agents

doi: 10.1002/1878-0261.13541

Figure Lengend Snippet: A similar extent of senescence induction by DNA‐damaging agents and CDK4/6i. (A) MCF‐7 cells were treated with DNA‐damaging agents (Cisplatin 5 μ m and Doxorubicin 100 n m ) and CDK4/6i (Palbociclib 2 μ m , Abemaciclib 1 μ m , and Ribociclib 5 μ m ) for 5 days and then SA‐β gal staining was performed. Representative images of SA‐β gal‐positive cells are shown. Scale bar, 100 μm. The percentage of stained cells was quantified and presented as a bar graph. Data are mean ± SD of four ( N = 4) independent experiments. The P ‐value was calculated using one‐way ANOVA. *** P < 0.001. (B) Cell cycle analysis of control, DNA‐damaging agents, and CDK4/6i‐treated senescent cells was performed by FACS. A representative bar graph shows the percentage of cells in each cell cycle phase. (C) The mRNA expression of genes was measured using RT‐qPCR in control, DNA‐damaging agents, and CDK4/6i‐induced senescent cells. The P ‐value was calculated by the one‐way ANOVA. Data are mean ± SD of three ( N = 3) independent experiments. * P < 0.05 and ** P < 0.01. (D, E) The cells were harvested after 5 days of drug treatment and subjected to immunoblotting for protein expression analysis. β‐actin and α‐tubulin were used as loading controls. Data are representative of three ( N = 3) independent experiments.

Article Snippet: DNA‐damaging agents including Doxorubicin, Cisplatin, Etoposide, and Carboplatin, CDK4/6i including Palbociclib, Abemaciclib, and Ribociclib, and aromatase inhibitors (AI) including Anastrozole, Letrozole (Selleck, Houston, TX) were treated to the cells.

Techniques: Staining, Cell Cycle Assay, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Oncology

Article Title: CDK4/6 inhibitors induce breast cancer senescence with enhanced anti‐tumor immunogenic properties compared with DNA‐damaging agents

doi: 10.1002/1878-0261.13541

Figure Lengend Snippet: A similar extent of senescence induction by DNA‐damaging agents and CDK4/6i. (A) MCF‐7 cells were treated with DNA‐damaging agents (Cisplatin 5 μ m and Doxorubicin 100 n m ) and CDK4/6i (Palbociclib 2 μ m , Abemaciclib 1 μ m , and Ribociclib 5 μ m ) for 5 days and then SA‐β gal staining was performed. Representative images of SA‐β gal‐positive cells are shown. Scale bar, 100 μm. The percentage of stained cells was quantified and presented as a bar graph. Data are mean ± SD of four ( N = 4) independent experiments. The P ‐value was calculated using one‐way ANOVA. *** P < 0.001. (B) Cell cycle analysis of control, DNA‐damaging agents, and CDK4/6i‐treated senescent cells was performed by FACS. A representative bar graph shows the percentage of cells in each cell cycle phase. (C) The mRNA expression of genes was measured using RT‐qPCR in control, DNA‐damaging agents, and CDK4/6i‐induced senescent cells. The P ‐value was calculated by the one‐way ANOVA. Data are mean ± SD of three ( N = 3) independent experiments. * P < 0.05 and ** P < 0.01. (D, E) The cells were harvested after 5 days of drug treatment and subjected to immunoblotting for protein expression analysis. β‐actin and α‐tubulin were used as loading controls. Data are representative of three ( N = 3) independent experiments.

Article Snippet: DNA‐damaging agents including Doxorubicin, Cisplatin, Etoposide, and Carboplatin, CDK4/6i including Palbociclib, Abemaciclib, and Ribociclib, and aromatase inhibitors (AI) including Anastrozole, Letrozole (Selleck, Houston, TX) were treated to the cells.

Techniques: Staining, Cell Cycle Assay, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Oncology

Article Title: CDK4/6 inhibitors induce breast cancer senescence with enhanced anti‐tumor immunogenic properties compared with DNA‐damaging agents

doi: 10.1002/1878-0261.13541

Figure Lengend Snippet: Differential expression of genes related to pro‐inflammatory response, anti‐tumor immunity, and angiogenesis induced by DNA‐damaging agents or CDK4/6i. (A) MCF‐7 cells were treated with DNA‐damaging agents (Cisplatin 5 μ m and Doxorubicin 100 n m ) and CDK4/6i (Palbociclib 2 μ m ) and RNA‐seq analysis was performed after 5 days. A heatmap was generated from the whole transcriptome of control and senescent cells, showing a hierarchical clustering correlation with a z ‐score between the samples. Data are mean ± SD of three ( N = 3) independent experiments. (B) Overlapping GSEA plots showing representative senescent gene sets including senescence, cell cycle, and DNA repair, with ES, in TIS compared to the control. (C) A similarity index matrix of the whole transcriptome comparing TIS and control. The color bar indicates the z ‐score range, from the row min value to the row max value. (D) A Venn diagram showing significantly up‐ and downregulated genes between TIS groups. Light green: Cis vs. Palbo; sky blue: Doxo vs. Palbo; and purple: Doxo vs. Cis. (E) GO analysis of the whole transcriptome in TIS, presented as a heatmap. Significantly enriched top 10 gene sets indicated with an asterisk. (F) NES of HALLMARK gene sets in TIS are shown as a bar graph. Significantly enriched top 10 gene sets are indicated with an asterisk. (G) Heatmaps showing the leading edge of HALLMARK gene sets with z ‐score in the whole transcriptome. Cis, Cisplatin; Doxo, Doxorubicin, Palbo, Palbociclib.

Article Snippet: DNA‐damaging agents including Doxorubicin, Cisplatin, Etoposide, and Carboplatin, CDK4/6i including Palbociclib, Abemaciclib, and Ribociclib, and aromatase inhibitors (AI) including Anastrozole, Letrozole (Selleck, Houston, TX) were treated to the cells.

Techniques: Quantitative Proteomics, RNA Sequencing, Generated, Control

Journal: Molecular Oncology

Article Title: CDK4/6 inhibitors induce breast cancer senescence with enhanced anti‐tumor immunogenic properties compared with DNA‐damaging agents

doi: 10.1002/1878-0261.13541

Figure Lengend Snippet: Differential expression of pro‐inflammatory SASP and ligands in DNA‐damaging agent‐induced senescent cancer cells compared to CDK4/6i‐induced senescent cancer cells. (A) A comparison of enrichment score (ES) of pro‐tumorigenic‐related gene sets among TIS groups presented as a dotplot with P ‐value; Cis vs. Palbo, Doxo vs. Palbo, and Doxo vs. Cis. (B) A cnetplot of the gene sets selected from (A), showing significantly upregulated genes (log 2 FC ≥0.5, P ≤ 0.05) compared between the TIS groups; sky blue: Cis vs. Palbo; purple: Doxo vs. Palbo; and light purple: Doxo vs. Cis. The labeled genes on the cnetplot indicate pro‐tumorigenic secretome, ligands, and cytokines belonging to each gene set. (C) An overlapping GSEA presenting ES (upper panel), rank in gene list (middle panel) between TIS vs. control. Indicated genes as black bars in the middle panel arranged in tabulated form based on their rank. The green gradient indicates the ranks of the genes (bottom panel). (D) A heatmap of pro‐tumorigenic immune response‐related SASP expression, generated from the transcriptome and presented with z ‐scores. The left panels of the heatmap indicate gene categories related to pro‐tumorigenic immune response, and the gray in the heatmap indicates unexpressed genes. (E) MCF‐7 cells were exposed to the DNA‐damaging agents and CDK4/6i and harvested after 5 days. The expression levels of mRNA encoding representative SASP, chemokines, and ligands related to pro‐tumorigenic immune responses were determined by RT‐qPCR. Data are mean ± SD of at least three ( N ≥ 3) independent experiments. The P ‐value was calculated using one‐way ANOVA. * P < 0.05 (TIS vs. Control) and # P < 0.05 (Cis vs. Doxo, Palbo vs. Doxo). (F) The cells (4 × 10 5 cells/100 mm dish) were treated and harvested after 5 days. The concentrations (pg·mL −1 ) of IL‐6 and CXCL8 were measured by ELISA in conditioned media collected from senescent and control cells. Data are mean ± SD of three ( N = 3) independent experiments. The P ‐value was calculated using one‐way ANOVA. * P < 0.05 (TIS vs. Control) and # P < 0.05 (Cis vs. Doxo, Palbo vs. Doxo). Cis, Cisplatin; Doxo, Doxorubicin; Palbo, Palbociclib.

Article Snippet: DNA‐damaging agents including Doxorubicin, Cisplatin, Etoposide, and Carboplatin, CDK4/6i including Palbociclib, Abemaciclib, and Ribociclib, and aromatase inhibitors (AI) including Anastrozole, Letrozole (Selleck, Houston, TX) were treated to the cells.

Techniques: Quantitative Proteomics, Comparison, Labeling, Control, Expressing, Generated, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: CDK4/6 inhibitors induce breast cancer senescence with enhanced anti‐tumor immunogenic properties compared with DNA‐damaging agents

doi: 10.1002/1878-0261.13541

Figure Lengend Snippet: Pro‐angiogenic and anti‐angiogenic factors are enriched in DNA‐damaging agent‐ and CDK4/6i‐induced senescent cells, respectively. (A) A comparison of enrichment score (ES) of pro‐angiogenic gene sets among TIS groups presented as a dotplot with P ‐value; Cis vs. Palbo, Doxo vs. Palbo, and Doxo vs. Cis. (B) A cnetplot of the gene sets selected from (A), showing a comparison of the significantly upregulated genes (log 2 FC ≥0.5, P ≤ 0.05) between TIS groups; sky blue: Cis vs. Palbo; purple: Doxo vs. Palbo; and light purple: Doxo vs. Cis. The labeled genes on the cnetplot indicate angiogenic factors belonging to each gene set. (C) An overlapping GSEA showing ES (upper panel) and rank in gene list (middle panel) between TIS vs. Control. Indicated genes are shown as black bars (middle panel) arranged in tabulated form based on rank. The green gradient indicates the ranks of the genes (bottom panel). (D) A heatmap of angiogenic factors was obtained from the transcriptome and presented as z ‐scores. The left panels of the heatmap indicate categories of pro‐ and anti‐angiogenic factors, and the gray in the heatmap indicates unexpressed genes. (E) MCF‐7 cells were treated with the agents and harvested after 5 days. The expression levels of pro‐angiogenic and anti‐angiogenic genes were determined by RT‐qPCR. Data are mean ± SD of at least three ( N = 3) independent experiments. The P ‐value was calculated using one‐way ANOVA. * P < 0.05 (TIS vs. Control) and # P < 0.05 (Cis vs. Doxo, Palbo vs. Doxo). (F) The cells (4 × 10 5 cells/100 mm dish) were treated and harvested after 5 days. The concentration (pg·mL −1 ) of VEGF was measured by ELISA in conditioned media collected from senescent and control cells. Data are mean ± SD of three ( N = 3) independent experiments. The P ‐value was calculated using one‐way ANOVA. * P < 0.05 (TIS vs. Control) and # P < 0.05 (Cis vs. Doxo, Palbo vs. Doxo). Cis, Cisplatin; Doxo, Doxorubicin; Palbo, Palbociclib.

Article Snippet: DNA‐damaging agents including Doxorubicin, Cisplatin, Etoposide, and Carboplatin, CDK4/6i including Palbociclib, Abemaciclib, and Ribociclib, and aromatase inhibitors (AI) including Anastrozole, Letrozole (Selleck, Houston, TX) were treated to the cells.

Techniques: Comparison, Labeling, Control, Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: CDK4/6 inhibitors induce breast cancer senescence with enhanced anti‐tumor immunogenic properties compared with DNA‐damaging agents

doi: 10.1002/1878-0261.13541

Figure Lengend Snippet: A similar antigen presentation and interferon signaling activities in DNA‐damaging agent‐ and CDK4/6i‐induced senescent cells. (A) A comparison of ES of anti‐tumorigenic immune response‐related gene sets among TIS groups with P ‐value; Cis vs. Palbo, Doxo vs. Palbo, and Doxo vs. Cis. (B) A cnetplot of the gene sets selected from (A), showing a comparison of the significantly upregulated genes (log 2 FC ≥0.5, P ≤ 0.05) of TIS groups vs. Control; orange: Cis vs. Control; red: Doxo vs. Control; and blue: Doxo vs. Control. The labeled genes in the cnetplot indicate genes associated with antigen presentation within each gene set. (C) An overlapping GSEA showing ES (upper panel), rank in gene list (middle panel) between TIS vs. Control. Indicated genes as black bars (middle panel) arranged in tabulated form based on their rank. The green gradient indicates the ranks of the gens (bottom panel). (D) A heatmap of anti‐tumorigenic immune response factors was obtained from the transcriptome and presented as z ‐scores. The left panels of the heatmap indicate the categories of anti‐tumorigenic immune response factors, and the gray in the heatmap indicates unexpressed genes. (E) MCF‐7 cells were treated with the agents and harvested after 5 days. The expression levels of anti‐tumorigenic were determined by RT‐qPCR. Data are mean ± SD of three ( N = 3) independent experiments. The P ‐value was calculated by the one‐way ANOVA. * P < 0.05 (TIS vs. Control) and # P < 0.05 (Cis vs. Doxo, Palbo vs. Doxo). (F, G) The cells (4 × 10 5 cells/100 mm dish) were treated and harvested after 5 days. The concentrations (pg·mL −1 ) of CXCL10 and IFN‐λ were measured by ELISA in conditioned media collected from senescent and control cells. Data are mean ± SD of three ( N = 3) independent experiments. The P ‐value was calculated using one‐way ANOVA. * P < 0.05 (TIS vs. Control) and # P < 0.05 (Cis vs. Doxo, Palbo vs. Doxo). Cis, Cisplatin; Doxo, Doxorubicin; Palbo, Palbociclib.

Article Snippet: DNA‐damaging agents including Doxorubicin, Cisplatin, Etoposide, and Carboplatin, CDK4/6i including Palbociclib, Abemaciclib, and Ribociclib, and aromatase inhibitors (AI) including Anastrozole, Letrozole (Selleck, Houston, TX) were treated to the cells.

Techniques: Immunopeptidomics, Comparison, Control, Labeling, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: CDK4/6 inhibitors induce breast cancer senescence with enhanced anti‐tumor immunogenic properties compared with DNA‐damaging agents

doi: 10.1002/1878-0261.13541

Figure Lengend Snippet: Exclusive activation of p53 and NF‐κB signaling in DNA‐damaging agent‐induced senescent cells. (A) IPA was performed to analyze upstream regulators and their expression levels were compared between TIS groups; Doxo vs. Palbo and Cis vs. Palbo. The top 10 regulators were ranked according to the z ‐score with P ≤ 0.05. (B) A comparison of ES of p53, NF‐κB, and ESR1 signaling‐related gene sets among TIS groups with P ‐value; Cis vs. Palbo, Doxo vs. Palbo, and Doxo vs. Cis. (C) MCF‐7 cells were treated with DNA‐damaging agents and CDK4/6i and harvested after 5 days. The p53, p21 Waf1/Cip1 expression, and NF‐κB activation were determined by immunoblotting. α‐tubulin was used as a loading control. Data are representative of three ( N = 3) independent experiments. (D) An illustration depicting the effect of DNA‐damaging agents and CDK4/6i‐induced senescence on the TME of breast cancer cells. Cis, Cisplatin; Doxo, Doxorubicin; Palbo, Palbociclib.

Article Snippet: DNA‐damaging agents including Doxorubicin, Cisplatin, Etoposide, and Carboplatin, CDK4/6i including Palbociclib, Abemaciclib, and Ribociclib, and aromatase inhibitors (AI) including Anastrozole, Letrozole (Selleck, Houston, TX) were treated to the cells.

Techniques: Activation Assay, Expressing, Comparison, Western Blot, Control