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Abstract
Purpose
Breast cancer has been reported as the most common cancer of women in the United States, Western Europe and Korea and about 210,000 and 10,000 women in United States and Korea every year, respectively are diagnosed with it. Breast cancer is curable with an early diagnosis, and many researchers have made efforts to find a marker for this malady, heat shock protein (HSP) consists of 6 groups, it is highly preserved throughout both the prokaryotic and eukaryotic cells and it acts as a molecular chaperone that's involved in protein folding. HSPs have been recently reported to be related with breast cancer. In this study, we investigated the changes of expression of HSP60 in tissues and cell lines of breast cancer.
Methods
We obtained breast cancer tissues and normal tissues from breast cancer patients, and we purchased several cancer cell lines from American tissue culture correction. We treated the tissues and the cell lines of human breast cancer with heat shock protein. Proteins and mRNAs were isolated from the tissues and the cells and then we performed Western blotting, reverse transcriptase-Polymerase chain Reaction and fluorescence activated cell sorter analysis on them.
Results
On Western blot, HSP60 was more overexpressed in the tissues and the cell lines of breast cancer than in the normal breast tissues and cell lines. The expression of HSP60 showed 2 types of molecular weight differences in the tissues and cell lines of breast cancer, and specifically, low HSP60 was over-expressed in the cancer tissues. There was no difference between the breast cancer cell lines and the normal cell lines in the expressions of HSP60 mRNA, according to the treatment with heat shock. Also, there was no relationship between phosphorylation and the structural difference of HSP60 protein, according to HSP60 protein's molecular weight. The expression of HSP60 has been mostly reported at the mitochondria; however, in this study, it was more predominantly detected at the cytoplasm than at the mitochondria in the breast cancer cell lines.
Figures and Tables
Fig 1
Structure of heat shock protein 60 kDa (HSP60). Molecular weight is 60 kDa, synthesized in the cytoplasm and transported to mitochondria.
Fig 2
Expression of HSP60 in cell line and tissue by western blot. Lane 1=cancer cell line (37℃); lane 2=cancer cell line (41℃ heat shock treatment); lane 3=cancer cell line (45℃ heat shock treatment); lane 4=normal cell line (37℃); lane 5=normal cell line (41℃); lane 6=breast cancer tissue; lane 7=breast normal tissue; lane 8=cervix cancer tissue; lane 9=cervix normal tissue.
Fig 3
Expression of HSP60 in MDA-MB-231 by western blot. HSP60 was detected by western blotting using heat shock 60 kDa protein 1 (chaperonin) polyclonal antibody. (A) Expression pattern of HSP60 in MDA-MB-231 cell line treated heat shock (37, 39, 41, 43, 45℃). (B) Expression pattern of β-actin in MDA-MB-231 cell line treated heat shock (37, 39, 41, 43, 45℃).
BC=Breast Cancer tissue; BN=Breast Normal tissue.
Fig 4
Expression of HSP60 in MCF-7 by western blot. HSP60 was detected by western blotting using heat shock 60 kDa protein 1 (chaperonin) polyclonal antibody. (A) Expression pattern of HSP60 in MCF-7 cell line treated heat shock (37, 39, 41, 43, 45℃). (B) Expression pattern of β-actin in MCF-7 cell line treated heat shock (37, 39, 41, 43, 45℃),
BC=Breast Cancer tissue; BN=Breast Normal tissue.
Fig 5
Expression of HSP60 in CCD-1113 by western blot. HSP60 was detected by western blotting using heat shock 60 kDa protein 1 (chaperonin) polyclonal antibody. (A) Expression pattern of HSP60 in CCD-1113sk cell line treated heat shock (37, 39, 41, 43, 45℃). (B) Expression pattern of β-actin in CCD-1113sk cell line treated heat shock (37, 39, 41, 43, 45℃).
BC=Breast Cancer tissue; BN=Breast Normal tissue.
Fig 6
Amplification of HSP60 and GAPDH in MDA-MB-231 by RT-PCR. A 2.5% agarose gel electrophoresis of RT-PCR products of MDA-MB-231 cell line mRNA. RT-PCR was carried out using the primer pair HSP60 and GAPDH. Lane M.W, size marker (phix174/BsuRI [HaeIII]). (A) RT-PCR products of HSP60 in MDA-MB-231 cell line treated with heat shock (37, 39, 41, 43, 45℃). (B) RT-PCR products of GAPDH in MDA-MB-231 cell line treated with heat shock (37, 39, 41, 43, 45℃).
BC=Breast Cancer tissue; BN=Breast Normal tissue.
Fig 7
Amplification of HSP60 and GAPDH in MCF-7 cell line by RT-PCR. A 2.5% agarose gel electrophoresis of RT-PCR products of MCF-7 cell line mRNA. RT-PCR was carried out using the primer pair HSP60 and GAPDH. Lane M.W, size marker (phix174/BsuRI [HaeIII]). (A) RT-PCR products of HSP60 in MCF-7 cell line treated with heat shock (37, 39, 41, 43, 45℃). (B) RT-PCR products of GAPDH in MCF-7 cell line treated with heat shock (37, 39, 41, 43, 45℃).
BC=Breast Cancer tissue; BN=Breast Normal tissue.
Fig 8
Amplification of HSP60 and GAPDH in CCD-1113 by RT-PCR. A 2.5% agarose gel electrophoresis of RT-PCR products of CCD-1113sk cell line mRNA. RT-PCR was carried out using the primer pair HSP60 and GAPDH. Lane M.W, size marker (phix174/BsuRI [HaeIII]). (A) RT-PCR products of HSP60 in CCD-1113sk cell line treated heat shock (37, 39, 41, 43, 45℃). (B) RT-PCR products of GAPDH in CCD-1113sk cell line treated heat shock (37, 39, 41, 43, 45℃).
BC=Breast Cancer tissue; BN=Breast Normal tissue.
Fig 9
Treatment of alkaline phosphatase (AP). (A) Expression of HSP60 in MDA-MB-231 cell line treated heat shock (37, 39, 41, 43, 45℃) by western blot. (B) Before alkaline phosphatase treatment. Lane 1=cancer cell line (37℃); lane 2=cancer cell line (41℃ heat shock treatment); lane 3=cancer cell line (45℃ heat shock treatment); lane 4=normal cell line (37℃); lane 5=normal cell line (41℃); lane 6=breast cancer tissue; lane 7=breast normal tissue. (C) After Alkaline Phosphatase treatement. Lane 1=cancer cell line (37℃); lane 2=cancer cell line (41℃ heat shock treatment); lane 3; cancer cell line (45℃ heat shock treatment); lane 4=normal cell line (37℃); lane 5=normal cell line (41℃); lane 6=breast cancer tissue; lane 7=breast normal tissue.
Fig 10
Extraction of cell membrane, nucleus and mitochondria. Lane 1=Nucleus protein extracted in cancer cell line; lane 2=Mitochondria protein extracted in cancer cell line; lane 3=cancer cell line; lane 4=normal cell line; lane 5=breast cancer tissue; lane 6=breast normal tissue.
Fig 11
Cytofluorimetric analysis of Mitofluor in living cell. FACS can analysis was performed with an direct method. Samples were acquired using a flow cytometer and data were analyzed with the Lysis II software (FACScan, Becton-Dickinson). (A) Normal, (B) Cancer, (C) compare cancer with normal cell line.
Fig 12
Expression of HSP60 and β-actin in Breast cancer and normal tissue by western blot. HSP60 was detected by western blotting using heat shock 60 kDa protein 1 (chaperonin) polyclonal antibody. (A) Expression pattern of HSP60 in breast cancer (1-6, 10-14, 18-26) and normal tissues (7-9, 15-17). (B) Expression pattern of β-actin in breast cancer (1-6, 10-14, 18-26) and normal tissues (7-9, 15-17).
Fig 13
Expression ratio of HSP60 and β-actin in Breast cancer and normal tissue by western blot. HSP60 was detected by western blotting using heat shock 60 kDa protein 1 (chaperonin) polyclonal antibody and β-actin antibody. (A) Expression pattern of HSP60 in breast normal and cancer tissues and (B) is β-actin (ANOVA t test [Mann Whitney], p<0.0001).
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