MADD knock-down enhances doxorubicin and TRAIL induced apoptosis in breast cancer cells

Abstract

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

Expression of MADD protein in breast cancer tissues.

A. Tissue microarrays (TMA) containing tissue sections representing benign breast lesions, ductal carcinoma in situ (DCIS) and invasive breast carcinomas (IBC) were prepared and stained for MADD expression. B. The TMAs were scored for the degree of MADD expression by two independent investigators in a semi-quantitative fashion (0 = negative, 1 = weak intensity, 2 = moderate intensity, 3 = strong intensity). C. Statistical analysis was carried out using one-way ANOVA with Tukey-Kramer post-hoc as described under materials and methods. A significant difference in the intensity of MADD stain in DCIS and IBC cases as compared to normal tissues was noted (p = 0.01 and p = 0.001 respectively).

Figure 2. Expression of MADD, and selective knock down of IG20 isoforms in breast cancer cells.

A. shows immunofluorescence staining of MADD in MCF-7, MDA-MB-231 and T47D breast cancer cell lines. B. Selective knockdown of IG20 isoforms in breast cancer cell lines. Our previously generated 16E and 13L-shRNA lentivirus can efficiently knock down the appropriate isoforms in MCF-7, MDA-MB-231 and T47D cells. All three cell lines were transduced with the indicated virus for 72 h at which point RNA was extracted and used (1μg) for RT-PCR using F2-B2 primers. The products were run on a 2% agarose gel to separate and visualize the isoforms.

Figure 3. MADD knockdown in breast cancer cells results in spontaneous apoptosis.

MCF-7 (A), MDA-MB-231(B) and T47D(C) breast cancer cells were transduced for 72 h with indicated viruses, they were then stained with TMRM, harvested and subjected to flow cytometry to determine the percentage of apoptotic cells. *p<0.05;** p<0.01 13L shRNA vs. 16E shRNA. Summarized data from three independent experiments are shown.

Figure 4. Upon MADD knockdown, low doses of TRAIL and doxorubicin can enhance apoptosis.

MCF-7 (A) and MDA-MB-231 (B) cells were treated with TRAIL for 24 h and with doxorubicin for 72 h at a concentration of 10 ng/ml. Cells were stained with TMRM and analyzed by FACS. Statistical significance levels are shown on the figures. Summarized data from three independent experiments are shown.

Figure 5. MADD Knockdown, followed by treatment with TRAIL or doxorubicin, results in increased caspase-8 activation.

MADD knockdown in combination with either TRAIL or doxorubicin results in extrinsic apoptosis. MCF-7 cells were transfected with either empty vector (pcDNA) or pcDNA-DN-FADD plasmid, Twenty four hours later the cells were transduced with 16E or 13L lentivirus for 72 hours and were left alone, or treated with TRAIL or doxorubicin. One third of the cells were stained with TMRM and subjected to FACS analysis to determine apoptosis (A), and the other two thirds were used for DR5 immunoprecipitation (B). Separated immune complexes were immunoblotted using antibodies specific for cleaved caspase-8 and DR5. Summarized data from three independent experiments are shown.