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Mammaglobin as a potential molecular target for breast cancer drug delivery.

Zuo L, Li L, Wang Q, Fleming TP, You S - Cancer Cell Int. (2009)

Bottom Line: Recently, several groups of researchers proposed a number of therapeutic strategies targeting this molecule.We provided several evidences to demonstrate the presence of the membrane-associated MAM.To test whether the membrane-associated MAM can serve as a molecular target for drug delivery, we conjugated anti-MAM antibody to human low-density lipoprotein (LDL) and loaded doxorubicin (Dox) in the core of LDL.

View Article: PubMed Central - HTML - PubMed

Affiliation: Atlanta Research and Education Foundation, Atlanta VA Medical Center (151), Decatur, GA 30033, USA. arefzuo@gmail.com

ABSTRACT

Background: Mammaglobin (MAM) has been used as a specific molecular marker for breast cancer diagnosis. Recently, several groups of researchers proposed a number of therapeutic strategies targeting this molecule. Some of the strategies are based upon an essential but not demonstrated hypothesis - mammaglobin is associated with the surface of breast cancer cells, which strongly disputes the therapeutic strategies.

Results: We conducted a computer-based predictive analysis and identified a small fragment at the N-end of MAM as a potential transmembrane domain. We provided several evidences to demonstrate the presence of the membrane-associated MAM. We isolated the membrane protein components from known MAM positive breast cancer cells (MDA-MB361 and MDA-MB415). We showed that about 22-64% of MAM proteins, depending upon the types of the cancer cells, directly attached on the membrane of breast cancer cells, by Western blotting assays. To directly visualize the presence of the membrane-bound MAM protein, we incubated the MAM positive cancer cells with FITC labeled anti-MAM antibody, and observed clear fluorescent signals on the surface of the cells. In studying the MAM protein distribution in human breast cancer tissues, we first identified two immunostain patterns that are associated with the membrane-bound MAM: the membrane stain pattern and luminary surface stain pattern. To test whether the membrane-associated MAM can serve as a molecular target for drug delivery, we conjugated anti-MAM antibody to human low-density lipoprotein (LDL) and loaded doxorubicin (Dox) in the core of LDL. Specific binding and cytotoxicity of the MAM targeted and Dox loaded LDL was tested in the MAM positive breast cancer cells in vitro.

Conclusion: We first showed that some of MAM protein directly associated with the surface of breast cancer cells. The membrane-associated MAM protein may be utilized as a useful molecular marker for breast cancer targeted drug delivery.

No MeSH data available.


Related in: MedlinePlus

Panel One: Incubation of MB361 cells with anti-MAM antibody and induction of cell apoptosis. Cell viability assay was conducted after incubation of MB361 breast cancer cells with anti-MAM. The number of the dead cells (in red color) was found higher in the anti-MAM incubated cancer cells (B), as compared with that in the non-incubated MB361 cells (A). There were no or few dead HAEC cells (C). The images were taken with objective lens 10×. Panel Two: Western blot assay for the pro-PARP-1 (100 kDa) and activated PARP-1 (89 kDa) in the protein lysates of MDA-MB361, T47D, and HAEC cells. The Con – control cells with no anti-MAM antibody inoculation, MAM – cells with anti-MAM antibody inoculation.
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Figure 4: Panel One: Incubation of MB361 cells with anti-MAM antibody and induction of cell apoptosis. Cell viability assay was conducted after incubation of MB361 breast cancer cells with anti-MAM. The number of the dead cells (in red color) was found higher in the anti-MAM incubated cancer cells (B), as compared with that in the non-incubated MB361 cells (A). There were no or few dead HAEC cells (C). The images were taken with objective lens 10×. Panel Two: Western blot assay for the pro-PARP-1 (100 kDa) and activated PARP-1 (89 kDa) in the protein lysates of MDA-MB361, T47D, and HAEC cells. The Con – control cells with no anti-MAM antibody inoculation, MAM – cells with anti-MAM antibody inoculation.

Mentions: After the incubation with 150 ng/ml of anti-MAM antibody, about 30–40% of MB361 cancer cells were detached from the cell culture slides. We assumed the cell apoptosis is induced. To test this assumption, we incubated MB361 cells with anti-MAM antibody. After 24 hour incubation, we conducted cell viability assays and found only about 10% of the attached cells dead (Figure 4 Panel One). In addition, we performed western blot assays to examine the expression of cell apoptosis relevant proteins under this condition from the cultured MB361, T47D, and HAEC cells. PARP-1 is a marker protein for cell apoptosis. The pro-PARP-1 protein is about 100 kDa and the activated form is 89 kDa. The activated PARP-1 was detected only in the MB361 cells incubated with anti-MAM antibody (Figure 4 Panel Two). However, there were no changes in the expression of other apoptosis relevant proteins such as caspase 3 and Bax-1 (data not shown) in the MB361 cells. These data indicated that only limited cell apoptosis was induced with the antibody incubation. The mechanisms that are involved in the cell detachment remain unclear.


Mammaglobin as a potential molecular target for breast cancer drug delivery.

Zuo L, Li L, Wang Q, Fleming TP, You S - Cancer Cell Int. (2009)

Panel One: Incubation of MB361 cells with anti-MAM antibody and induction of cell apoptosis. Cell viability assay was conducted after incubation of MB361 breast cancer cells with anti-MAM. The number of the dead cells (in red color) was found higher in the anti-MAM incubated cancer cells (B), as compared with that in the non-incubated MB361 cells (A). There were no or few dead HAEC cells (C). The images were taken with objective lens 10×. Panel Two: Western blot assay for the pro-PARP-1 (100 kDa) and activated PARP-1 (89 kDa) in the protein lysates of MDA-MB361, T47D, and HAEC cells. The Con – control cells with no anti-MAM antibody inoculation, MAM – cells with anti-MAM antibody inoculation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662795&req=5

Figure 4: Panel One: Incubation of MB361 cells with anti-MAM antibody and induction of cell apoptosis. Cell viability assay was conducted after incubation of MB361 breast cancer cells with anti-MAM. The number of the dead cells (in red color) was found higher in the anti-MAM incubated cancer cells (B), as compared with that in the non-incubated MB361 cells (A). There were no or few dead HAEC cells (C). The images were taken with objective lens 10×. Panel Two: Western blot assay for the pro-PARP-1 (100 kDa) and activated PARP-1 (89 kDa) in the protein lysates of MDA-MB361, T47D, and HAEC cells. The Con – control cells with no anti-MAM antibody inoculation, MAM – cells with anti-MAM antibody inoculation.
Mentions: After the incubation with 150 ng/ml of anti-MAM antibody, about 30–40% of MB361 cancer cells were detached from the cell culture slides. We assumed the cell apoptosis is induced. To test this assumption, we incubated MB361 cells with anti-MAM antibody. After 24 hour incubation, we conducted cell viability assays and found only about 10% of the attached cells dead (Figure 4 Panel One). In addition, we performed western blot assays to examine the expression of cell apoptosis relevant proteins under this condition from the cultured MB361, T47D, and HAEC cells. PARP-1 is a marker protein for cell apoptosis. The pro-PARP-1 protein is about 100 kDa and the activated form is 89 kDa. The activated PARP-1 was detected only in the MB361 cells incubated with anti-MAM antibody (Figure 4 Panel Two). However, there were no changes in the expression of other apoptosis relevant proteins such as caspase 3 and Bax-1 (data not shown) in the MB361 cells. These data indicated that only limited cell apoptosis was induced with the antibody incubation. The mechanisms that are involved in the cell detachment remain unclear.

Bottom Line: Recently, several groups of researchers proposed a number of therapeutic strategies targeting this molecule.We provided several evidences to demonstrate the presence of the membrane-associated MAM.To test whether the membrane-associated MAM can serve as a molecular target for drug delivery, we conjugated anti-MAM antibody to human low-density lipoprotein (LDL) and loaded doxorubicin (Dox) in the core of LDL.

View Article: PubMed Central - HTML - PubMed

Affiliation: Atlanta Research and Education Foundation, Atlanta VA Medical Center (151), Decatur, GA 30033, USA. arefzuo@gmail.com

ABSTRACT

Background: Mammaglobin (MAM) has been used as a specific molecular marker for breast cancer diagnosis. Recently, several groups of researchers proposed a number of therapeutic strategies targeting this molecule. Some of the strategies are based upon an essential but not demonstrated hypothesis - mammaglobin is associated with the surface of breast cancer cells, which strongly disputes the therapeutic strategies.

Results: We conducted a computer-based predictive analysis and identified a small fragment at the N-end of MAM as a potential transmembrane domain. We provided several evidences to demonstrate the presence of the membrane-associated MAM. We isolated the membrane protein components from known MAM positive breast cancer cells (MDA-MB361 and MDA-MB415). We showed that about 22-64% of MAM proteins, depending upon the types of the cancer cells, directly attached on the membrane of breast cancer cells, by Western blotting assays. To directly visualize the presence of the membrane-bound MAM protein, we incubated the MAM positive cancer cells with FITC labeled anti-MAM antibody, and observed clear fluorescent signals on the surface of the cells. In studying the MAM protein distribution in human breast cancer tissues, we first identified two immunostain patterns that are associated with the membrane-bound MAM: the membrane stain pattern and luminary surface stain pattern. To test whether the membrane-associated MAM can serve as a molecular target for drug delivery, we conjugated anti-MAM antibody to human low-density lipoprotein (LDL) and loaded doxorubicin (Dox) in the core of LDL. Specific binding and cytotoxicity of the MAM targeted and Dox loaded LDL was tested in the MAM positive breast cancer cells in vitro.

Conclusion: We first showed that some of MAM protein directly associated with the surface of breast cancer cells. The membrane-associated MAM protein may be utilized as a useful molecular marker for breast cancer targeted drug delivery.

No MeSH data available.


Related in: MedlinePlus