Article Figures & Data
Figures
- Figure 1.
Nrf2 is depleted in breast cancer cell lines. Nrf2 protein and RNA levels in breast cancer cell lines were compared with HMECs. A, Western blotting for nuclear Nrf2 in MCF-7, MDA-231, and ZR-75 breast cancer cell lines. B, Nrf2 mRNA expression detected by RT-PCR on total RNA extracts. C, Western blotting for basal expression of GCL and GSTA1/2 in MCF-7, MDA-231, and ZR-75 breast cancer cell lines. GAPDH was used as a loading control.
- Figure 2.
Cul3 is overexpressed, and Keap 1 varies in expression in Nrf2 depleted breast cancer cell lines. Breast cancer cell lines with low Nrf2 levels were assessed for Cul3 and Keap1 protein levels compared with HMECs. A, Western blotting for total Cul3 or Keap1 in HMEC and in MCF-7, MDA-231, and ZR-75 breast cancer cell lines. B, Cul3 and Keap1 mRNA expression detected by RT-PCR on total RNA extracts from the same cell lines. GAPDH was used as a loading control.
- Figure 3.
Cul3 is overexpressed in Nrf2-depleted breast cancer tumors. In 7 of 10 samples, breast cancer tissue samples expressing low Nrf2 level and matching surrounding normal mammary tissues were stained by immunohistochemistry for Cul3. Representative photomicrographs of 10 samples. A, normal mammary tissue showing Cul3 expression. B, breast cancer tissue showing Cul3 overexpression. C, normal mammary tissue with the presence of Nrf2, compared with (D) cancer cells with significantly lower Nrf2 signal.
- Figure 4.
Nrf2 and phase I/II enzymes are induced in Cul3-silenced breast cancer cells. Cul3 was stably silenced in MCF-7 cells using two different siRNA retroviral constructs (Cul3.1 and Cul3.2) to two different polyclonal populations as described in Materials and Methods. The expression of Cul3, Nrf2, and the enzymes AKR1C1, GCL, and UGDH was determined in parental MCF7 cells (wild-type), MCF7 transduced with an empty retroviral construct (control), and the two siRNA Cul3 (Cul3.1 and Cul3.2). A, Western blotting for total Cul3 and Nrf2. B, Western blotting for nuclear AKR1C1 and GCL; GAPDH was used as a loading control. C, AKR1C1, GCL, and UGDH mRNA expression detected by RT-PCR on total RNA extracts. D, relative quantification of hABCC1transporter using Quantitative PCR.
- Figure 5.
Cul3-silenced MCF-7 cells are resistant to H2O2- and serum starvation–induced cytotoxicity. Cul3-silenced cells were compared with wild-type and control cells to examine the cytotoxicity of (A) H2O2, (B) BaP, (C) Doxorubicin, and (D) Paclitaxel. Cell survival curves with data from at least three separate experiments are shown; points, mean; bars, SD.
Tables
- Table 1.
List of primers used in RT-PCR
Gene Primer pairs (sequences 5′→3′) Forward Reverse Nrf2 TCACCATCTCAGGGGGAG CAACATACTGACACTCCAATGC Cul-3 AGAGCGGAAAGGAGAAGTCGTAGA CTCAAAGTCACCCGCAATAGTT Keap1 CAGCCAAGGTCCCTGAGT GTGTAGCTGAAGGTGCGGT γGCS AGAGAAGGGGGAAAGGACAA GTGAACCCAGGACAGCCTAA GSTP1 CCCTACACCGTGGTCTATTTCC GAGGCTTTGAGTGAGCCCT NQO1 TGAAGGACCCTGCGAACTTTC GAACACTCGCTCAAACCAGC AKR1C1 CAATTCCCATCGACCAGAGT TACTTGAGCCCTGGCTTGTT UGDH GATGGGCTCCACAATGAACT TGGGCAACAGTGAGACTCTG Thioredoxin CAACCCTTTCTTTCATTCCCTCT CACCCACCTTTTGTCCCTTCT - Table 2.
Phase I and phase II enzymes up-regulated in Cul3-silenced MCF-7 cells
Genebank accession no. Detoxifying enzyme Fold increase Function NM_014862 Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) 3.69 ± 0.27 Phase I NM_000104 Cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) 4.02 ± 0.45 Phase I NM_001353 Aldo-keto reductase family 1, member C1 (AKR1C1) 4.29 ± 0.791 Phase II NM_003774 Udp-n-acetyl-α- d-galactosamine: polypeptide N-acetylgalactosaminyltransferase 4 (GalNAc-T4) 4.15 ± 0.97 Phase II NM_199127 γ-Glutamyltransferase-like 4 (GGTL4) 2.67 ± 0.50 Phase II NM_003312 Thiosulfate sulfurtransferase (rhodanese; TST) 2.08 ± 0.55 Phase II NM_003329 Thioredoxin (TXN) 1.63 ± 0.10 Phase II NM_003359 UGDH 7.70 ± 0.43 Phase II
Volume 8, Issue 8
