| Genistein is a soy isoflavone with phytoestrogen and tyrosine kinase inhibitory properties. High intake of soy/genistein has been associated with reduced breast cancer risk. Despite the advances in genistein-mediated anti-tumor studies, the underlying mechanisms remain unclear. In this study, we investigated genistein-induced regulation of cancerous inhibitor of protein phosphatase 2A(CIP2A), a novel oncogene frequently overexpressed in breast cancer, and its functional impact on genistein-induced growth inhibition and apoptosis. We demonstrated that genistein induced downregulation of CIP2 A in MCF-7-caspase-3(MCF-7-C3) and T47 D breast cancer cells, which was correlated with its growth inhibition and apoptotic activities. Overexpression of CIP2 A attenuated, whereas knockdown of CIP2 A sensitized, genistein-induced growth inhibition and apoptosis. We further showed that genistein-induced downregulation of CIP2 A involved both transcriptional suppression and proteasomal degradation. In particular, genistein at higher concentrations induced concurrent downregulation of E2F1 and CIP2 A. Overexpression of E2F1 attenuated genistein-induced downregulation of CIP2 A m RNA, indicating the role of E2F1 in genistein-induced transcriptional suppression of CIP2 A. As an extension of CIP2 A and genistein studies, we also explored the role of CIP2 A in acquired resistance to the ErbB2-targeting drug, lapatinib. We also demonstrated that CIP2 A knockdown in lapatinib-resistant(LR) BT474 breast cancer cells sensitized these cells to lapatinib-induced growth inhibition and apoptosis. These data further supported CIP2 A as a key regulator in anti-cancer drug response and resistance.Taken together, our results identified CIP2 A as a functional target of genistein demonstrated that the modulation of E2F1-mediated transcriptional regulation of CIP2 A contributes to its downregulation. These data advance our understanding of genistein-induced growth inhibition and apoptosis, and support the investigation of CIP2 A as a therapeutic target of relevant anti-cancer agents. Our data also lay the foundation for further investigation of CIP2 A in lapatinib resistance.PART ? Genistein-mediated regulation of CIP2 A in breast cancer cellsObjective To determine whether genistein induces CIP2 A regulation, growth inhibition and apoptosis in MCF-7, MCF-7-C3 and T47 D cells.Methods 1. CIP2 A, PARP/cleaved PARP and caspase-3/cleaved caspase-3 protein levels were examined in genistein-treated MCF-7, MCF-7-caspase-3(MCF-7-C3) and T47 D breast cancer cells. 2. Under similar genistein treatment conditions, we analyzed the inhibition of cell proliferation in MCF-7-C3 and T47 D cells by MTT assay. 3. The proportion of MCF-7-C3 and T47 D cells in each phase of the cell cycle was detected by flow cytometry.Results 1. When the cells were treated with genistein at concentrations ranging from 0 to 60 ?M for 48 h, CIP2 A protein levels in each cell line were downregulated in a concentration-dependent manner, especially in the 30 and 60 ?M groups. Genistein-mediated CIP2 A downregulation was associated with growth inhibition and PARP cleavage in MCF-7-C3 and T47 D cells that express functional caspase-3. 2. Under similar treatment conditions, genistein inhibited cell proliferation, as measured by MTT assay, in a concentration-dependent manner. 3. Genistein induced cell cycle arrest in G0/G1 phase, as measured by flow cytometry, when compared to corresponding controls.Conclusion Genistein-mediated downregulation of CIP2 A may have a functional impact on its tumor inhibition. These results demonstrated that CIP2 A is a cellular target of genistein.PART ? CIP2 A overexpression reduces breast cancer cell sensitivity to genistein-mediated growth inhibition and apoptosisObjective To determine whether CIP2 A overexpression reduces breast cancer cell sensitivity to genistein-mediated growth inhibition and apoptosis. To study the role of CIP2 A in genistein-induced apoptosis.Methods 1. CIP2 A in MCF-7-C3 and T47 D cells were overexpressed and characterized their responses to genistein. By infecting the cells with control and CIP2A-encoding lentiviruses, followed by puromycin selection, we obtained control(MCF-7-C3/CON and T47D/CON), CIP2A-overexpressing(MCF-7-C3/CIP2 A and T47D/CIP2A) stable lines. We verified CIP2 A protein levels by Western blot. 2. The survival fraction of MCF-7-C3/CIP2 A, T47D/CIP2 A and corresponding control cells were analysed by MTT. 3. Cell cycle analysis, as measured by flow cytometry, was performed to determine the proportion of MCF-7-C3/CIP2 A, T47D/CIP2 A and corresponding control cells in each phase of the cell cycle. 4. ELISA assays measured genistein-induced apoptosis in MCF-7-C3/CIP2 A, T47D/CIP2 A and control cells. 5. CIP2 A and PARP cleavage were detected by Western blot in genistein-treated MCF-7-C3/CIP2 A, T47D/CIP2 A and control cells.Results 1. After puromycin selection, we obtained control(MCF-7-C3/CON and T47D/CON), CIP2A-overexpressing(MCF-7-C3/CIP2 A and T47D/CIP2A) stable lines. 2. Genistein-induced growth inhibition in MCF-7-C3/CIP2 A and T47D/CIP2 A cells was significantly attenuated as compared to corresponding control cells, indicating that CIP2 A is involved in genistein-associated anti-proliferative effects. 3. Cell cycle analysis using flow cytometry indicated that genistein-induced G0/G1 arrest in both MCF-7-C3/CIP2 A and T47D/CIP2 A cells was decreased as compared to the control cells. 4. Apoptosis ELISA assays showed that genistein-induced apoptosis in MCF-7-C3/CIP2 A and T47D/CIP2 A cells was significantly decreased as compared to MCF-7-C3/CON and T47D/CON cells. 5. PARP cleavages in genistein-treated MCF-7-C3/CIP2 A and T47D/CIP2 A cells were also decreased as compared to the control cells.Conclusion Overexpression of CIP2 A attenuates genistein-induced growth inhibition and renders breast cancer cells resistant to genistein-induced apoptosis.PART ? CIP2 A knockdown enhances breast cancer cell sensitivity to genistein-mediated growth inhibition and apoptosisObjective To further study the role of CIP2 A in genistein-induced apoptosis and determine whether CIP2 A knockdown enhances breast cancer cell sensitivity to genistein-mediated growth inhibition and apoptosis.Methods 1. CIP2 A in MCF-7-C3 and T47 D cells were knocked down and characterized their responses to genistein. By infecting the cells with control and CIP2 A sh RNA-encoding lentiviruses, followed by puromycin selection, we obtained control(MCF-7-C3/CON and T47D/CON) and CIP2A-knockdown(MCF-7-C3/si CIP2 A and T47D/si CIP2A) cells. 2. The survival fraction of MCF-7-C3/si CIP2 A, T47D/si CIP2 A and control cells were examined by MTT assay. 3. Cell cycle analysis, using flow cytometry, was performed to determine the proportion of MCF-7-C3/si CIP2 A, T47D/si CIP2 A and control cells in each phase of the cell cycle. 4. Genistein-induced apoptosis in MCF-7-C3/si CIP2 A, T47D/si CIP2 A and corresponding control cells was measured by ELISA assay. 5. Western blot analysis was used to detect CIP2 A and PARP cleavage in genistein-treated MCF-7-C3/si CIP2 A, T47D/si CIP2 A and control cells.Results 1. After puromycin selection, we obtained control(MCF-7-C3/CON and T47D/CON) and CIP2A-knockdown(MCF-7-C3/si CIP2 A and T47D/si CIP2A) stable cell lines. 2. Genistein-induced growth inhibition in MCF-7-C3/si CIP2 A and T47D/si CIP2 A cells was significantly enhanced as compared to control cells. 3. Cell cycle analysis using flow cytometry indicated that genistein-induced G0/G1 arrest in both MCF-7-C3/si CIP2 A and T47D/si CIP2 A cells was further enhanced as compared to control cells. 4. Apoptosis ELISA assays showed that genistein-induced apoptosis in MCF-7-C3/si CIP2 A and T47D/si CIP2 A cells was significantly increased as compared to MCF-7-C3/CON and T47D/CON cells. 5. PARP cleavage in genistein-treated MCF-7-C3/si CIP2 A and T47D/si CIP2 A cells was also increased as compared to the control cells.Conclusion CIP2 A knockdown enhances genistein-induced growth inhibition and apoptosis.PART ? The mechanism of genistein-induced CIP2 A downregulationObjective To understand the mechanism of genistein-induced CIP2 A downregulation by transcriptional regulation and proteasomal degradationMethods 1. CIP2 A m RNA levels were examined in genistein-treated MCF-7-C3 and T47 D cells by q PCR. 2. Whether modulation of proteasomal degradation contributes to genistein-mediated downregulation of CIP2 A using a proteasomal inhibitor MG132. 3. As CIP2 A protein stability could be regulated by Akt activation, we examined phospho-Akt levels in genistein-treated cells. E2F1 is a major transcription factor that regulates CIP2 A transcription; therefore, we examined the role of E2F1 in genistein-mediated transcriptional regulation of CIP2 A. 4. MCF-7-C3 and T47 D cells were infected with adenovirus E2F1 and then E2F1, CIP2 A and c-Myc protein expression was detected by western blot. 5. CIP2 A m RNA levels in cells were examined with different E2F1 statuses and genistein treatments.Results 1. q PCR results show that CIP2 A m RNA levels in the cells treated with 25 or ?M genistein were significantly decreased. However, CIP2 A m RNA levels in the treated with genistein at lower concentrations were slightly increased. This dose-specific pattern was consistent with the protein level changes observed above. These results indicate that genistein-mediated inhibition of CIP2 A transcription is involved in the downregulation of CIP2 A. 2. Western blot analysis shows that treating the cells with the proteasomal inhibitor, MG132, significantly reversed genistein-induced downregulation of CIP2 A, suggesting that genistein also regulates CIP2 A at the protein level. 3. Phospho-Akt levels in genistein-treated breast cancer cells were inhibited at the protein level. Inhibition of Akt activation in the treated cells suggests a link to CIP2 A protein degradation. 4. E2F1 overexpression attenuated genistein-induced downregulation of CIP2 A and c-Myc protein levels. 5. E2F1 overexpression dramatically increased CIP2 A m RNA, whereas genistein-induced downregulation of CIP2 A m RNA was abolished in both cell lines with E2F1 overexpression.Conclusion E2F1 plays a critical role in genistein-mediated downregulation of CIP2 A m RNA, which contributes to the overall downregulation of CIP2 A protein levels.PART ? The regulation of CIP2 A in lapatinib-resistant cellsObjective To detect the sensitive change to lapatinib-induced apoptosis after CIP2 A knockdown in BT474/LR(lapatinib-resistant) cells. To study CIP2 A regulation in lapatinib-resistant cells.Methods 1. The stable BT474 lapatinib-resistant(BT4747/LR) and BT474 cells were treated with lapatinib and then analyzed the cell survival responses to lapatinib by MTT. 2. CIP2 A levels in the BT474 and BT474/LR cells were dectected by Western blot after treated with lapatinib.3. CIP2 A in BT474/LR was knocked down by infecting the cells with control and CIP2 A sh RNA-encoding lentivirus, followed by puromycin selection, to obtain control and BT474/LR/si CIPA stable cells. 4. The survival fraction of BT474/LR and BT474/LR/si CIP2 A cells were examined, which were treated with lapatinib, by MTT assay. 5. Apoptosis was measured using ELISA assays of lapatinib-treated BT474/LR and BT474/LR/si CIP2 A cells. 6. Western blot analysis was used to detect CIP2 A, PARP, caspase-3 and cleaved caspase-3(c-caspase-3) levels in lapatinib-treated BT474/LR and BT474/LR/si CIP2 A cells. 7. Protein levels of CIP2 A, p-Akt and Akt in lapatinib-treated BT474/LR and BT474/LR/si CIP2 A cells were detected by Western blot.Results 1. BT474/LR cells significantly resisted lapatinib-induced downregulation of CIP2 A as compared to control cells. 2. After puromycin selection, we obtained control and BT474/LR/si CIP2 A stable lines, as verified by Western blot analysis of CIP2 A protein expression. 3. CIP2 A knockdown significantly sensitized BT474/LR cells to lapatinib-induced growth inhibition. 4. Lapatinib-induced apoptosis in BT474/LR/si CIP2 A was significantly increased as compared to BT474/LR cells. 5. We found that lapatinib increased PARP and caspase-3 cleavage in BT474/LR/si CIP2 A cells as compared to the control cells. 6. Lapatinib treatment in BT474/LR/si CIP2 A cells inhibited phospho-Akt protein levels. Inhibition of phospho-Akt in the treated cells suggests a link to CIP2 A protein degradation.Conclusion CIP2 A knockdown enhances lapatinib-induced growth inhibition and apoptosis in BT474/LR cells. The mechanism may relate to lapatinib-induced inhibition of p-Akt after CIP2 A knockdown in BT474/LR cells. |
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