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Knockdown of the Inhibitor of Apoptosis BRUCE Sensitizes Resistant Breast Cancer Cells to Chemotherapeutic Agents.

Garrison JB, Ge C, Che L, Pullum DA, Peng G, Khan S, Ben-Jonathan N, Wang J, Du C - J Cancer Sci Ther (2015)

Bottom Line: However, they are not effective to all patients and patients often develop resistance.Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone.Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, USA.

ABSTRACT

Background and objectives: Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles.

Results and methods: IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone.

Conclusions: Our data suggest that elevated protein levels of BRUCE in breast tumors may contribute to chemoresistance in breast cancer patients. In support of this suggestion, our data demonstrate that a reduction in BRUCE expression in breast cancer cell lines increases the toxicity of several chemotherapeutic agents. In all likelihood, the contribution of increased BRUCE levels to chemoresistance are likely due to its roles in suppression of apoptosis, promotion of cytokinesis and facilitation of DNA damage repair. These observations suggest that therapeutic suppression of BRUCE could improve chemosensitivity in chemo-resistant breast cancer patients. Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.

No MeSH data available.


Related in: MedlinePlus

BRUCE knockdown sensitizes resistant MDA-MB-231 cells to cisplatin and taxol-induced cell death(A) Western blot of BRUCE protein levels preand post-lentiviral knockdown for 48 hrs in MDA-MB-231 cells exposed to lentivirus expressing five BRUCE-targeted shRNA (#7 – #11) and a scramble control (ctrl). (B) MTT assay of MDA-MB-231, MDA-MB-468, and MCF-7 cell lines following 1–5 days of lentiviral exposure (shRNA #9). Result of three independent experiments +/− SEM. (C) MDA-MB-231 cells treated with control or shBRUCE lentivirus for two days were exposed to increasing concentrations of cisplatin (0–10 μM) for three days and cell viability was measured by MTT assay. (D) MDA-MB-231 cells were exposed to shBRUCE lentivirus for two days followed by treatment with cisplatin (5 μM) or taxol (0.65 μM) for the indicated time points. Following treatment, the MTT assay for cell viability was performed. (E) MDA-MB-231 cells exposed to shBRUCE lentivirus for two days were co-exposed to cisplatin (2.5 μM) and taxol (0.65 μM) for the indicated time points. Following treatment the MTT assay for cell viability was performed. Result of three independent experiments +/− SEM.
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Figure 3: BRUCE knockdown sensitizes resistant MDA-MB-231 cells to cisplatin and taxol-induced cell death(A) Western blot of BRUCE protein levels preand post-lentiviral knockdown for 48 hrs in MDA-MB-231 cells exposed to lentivirus expressing five BRUCE-targeted shRNA (#7 – #11) and a scramble control (ctrl). (B) MTT assay of MDA-MB-231, MDA-MB-468, and MCF-7 cell lines following 1–5 days of lentiviral exposure (shRNA #9). Result of three independent experiments +/− SEM. (C) MDA-MB-231 cells treated with control or shBRUCE lentivirus for two days were exposed to increasing concentrations of cisplatin (0–10 μM) for three days and cell viability was measured by MTT assay. (D) MDA-MB-231 cells were exposed to shBRUCE lentivirus for two days followed by treatment with cisplatin (5 μM) or taxol (0.65 μM) for the indicated time points. Following treatment, the MTT assay for cell viability was performed. (E) MDA-MB-231 cells exposed to shBRUCE lentivirus for two days were co-exposed to cisplatin (2.5 μM) and taxol (0.65 μM) for the indicated time points. Following treatment the MTT assay for cell viability was performed. Result of three independent experiments +/− SEM.

Mentions: With the aim to determine if knockdown of BRUCE sensitizes resistant breast cancer cells to chemotherapeutic drugs, we screened five different BRUCE-targeted lentiviral shRNAs for their effect on BRUCE knockdown in the three cell lines. Western blot results demonstrated that shRNAs #9 and #11 were most effective for knockdown of BRUCE after 2 days of lentiviral shRNA infection in all three cell lines as exemplified in MDA-MB-231 cells (Figure 3A). Therefore, shRNA #9 was used in the following experiment to assess the cell viability over a course of 5 days of lentivirus-mediated knockdown of BRUCE. Results of MTT viability assay demonstrated that all three cell lines still exhibited 100% viability post 2 days of shRNA treatment, however, BRUCE was already depleted at this time. The cell viability continued to remain at 100% on day 3, slightly dropped on day 4, and further reduced on day 5 (Figure 3B). These results indicate that loss of cell viability does not occur simultaneously with loss of BRUCE expression, but ensures two days afterwards. Thus, lentiviral shRNA treatment for 2 days, during which BRUCE knockdown has been achieved and cell viability is still well preserved, was used in the following experiments to test the effect of BRUCE depletion on cell sensitivity to chemotherapeutic drugs.


Knockdown of the Inhibitor of Apoptosis BRUCE Sensitizes Resistant Breast Cancer Cells to Chemotherapeutic Agents.

Garrison JB, Ge C, Che L, Pullum DA, Peng G, Khan S, Ben-Jonathan N, Wang J, Du C - J Cancer Sci Ther (2015)

BRUCE knockdown sensitizes resistant MDA-MB-231 cells to cisplatin and taxol-induced cell death(A) Western blot of BRUCE protein levels preand post-lentiviral knockdown for 48 hrs in MDA-MB-231 cells exposed to lentivirus expressing five BRUCE-targeted shRNA (#7 – #11) and a scramble control (ctrl). (B) MTT assay of MDA-MB-231, MDA-MB-468, and MCF-7 cell lines following 1–5 days of lentiviral exposure (shRNA #9). Result of three independent experiments +/− SEM. (C) MDA-MB-231 cells treated with control or shBRUCE lentivirus for two days were exposed to increasing concentrations of cisplatin (0–10 μM) for three days and cell viability was measured by MTT assay. (D) MDA-MB-231 cells were exposed to shBRUCE lentivirus for two days followed by treatment with cisplatin (5 μM) or taxol (0.65 μM) for the indicated time points. Following treatment, the MTT assay for cell viability was performed. (E) MDA-MB-231 cells exposed to shBRUCE lentivirus for two days were co-exposed to cisplatin (2.5 μM) and taxol (0.65 μM) for the indicated time points. Following treatment the MTT assay for cell viability was performed. Result of three independent experiments +/− SEM.
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Figure 3: BRUCE knockdown sensitizes resistant MDA-MB-231 cells to cisplatin and taxol-induced cell death(A) Western blot of BRUCE protein levels preand post-lentiviral knockdown for 48 hrs in MDA-MB-231 cells exposed to lentivirus expressing five BRUCE-targeted shRNA (#7 – #11) and a scramble control (ctrl). (B) MTT assay of MDA-MB-231, MDA-MB-468, and MCF-7 cell lines following 1–5 days of lentiviral exposure (shRNA #9). Result of three independent experiments +/− SEM. (C) MDA-MB-231 cells treated with control or shBRUCE lentivirus for two days were exposed to increasing concentrations of cisplatin (0–10 μM) for three days and cell viability was measured by MTT assay. (D) MDA-MB-231 cells were exposed to shBRUCE lentivirus for two days followed by treatment with cisplatin (5 μM) or taxol (0.65 μM) for the indicated time points. Following treatment, the MTT assay for cell viability was performed. (E) MDA-MB-231 cells exposed to shBRUCE lentivirus for two days were co-exposed to cisplatin (2.5 μM) and taxol (0.65 μM) for the indicated time points. Following treatment the MTT assay for cell viability was performed. Result of three independent experiments +/− SEM.
Mentions: With the aim to determine if knockdown of BRUCE sensitizes resistant breast cancer cells to chemotherapeutic drugs, we screened five different BRUCE-targeted lentiviral shRNAs for their effect on BRUCE knockdown in the three cell lines. Western blot results demonstrated that shRNAs #9 and #11 were most effective for knockdown of BRUCE after 2 days of lentiviral shRNA infection in all three cell lines as exemplified in MDA-MB-231 cells (Figure 3A). Therefore, shRNA #9 was used in the following experiment to assess the cell viability over a course of 5 days of lentivirus-mediated knockdown of BRUCE. Results of MTT viability assay demonstrated that all three cell lines still exhibited 100% viability post 2 days of shRNA treatment, however, BRUCE was already depleted at this time. The cell viability continued to remain at 100% on day 3, slightly dropped on day 4, and further reduced on day 5 (Figure 3B). These results indicate that loss of cell viability does not occur simultaneously with loss of BRUCE expression, but ensures two days afterwards. Thus, lentiviral shRNA treatment for 2 days, during which BRUCE knockdown has been achieved and cell viability is still well preserved, was used in the following experiments to test the effect of BRUCE depletion on cell sensitivity to chemotherapeutic drugs.

Bottom Line: However, they are not effective to all patients and patients often develop resistance.Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone.Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, USA.

ABSTRACT

Background and objectives: Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles.

Results and methods: IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone.

Conclusions: Our data suggest that elevated protein levels of BRUCE in breast tumors may contribute to chemoresistance in breast cancer patients. In support of this suggestion, our data demonstrate that a reduction in BRUCE expression in breast cancer cell lines increases the toxicity of several chemotherapeutic agents. In all likelihood, the contribution of increased BRUCE levels to chemoresistance are likely due to its roles in suppression of apoptosis, promotion of cytokinesis and facilitation of DNA damage repair. These observations suggest that therapeutic suppression of BRUCE could improve chemosensitivity in chemo-resistant breast cancer patients. Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.

No MeSH data available.


Related in: MedlinePlus