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In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance.

Wang W, Yin L, Gonzalez-Malerva L, Wang S, Yu X, Eaton S, Zhang S, Chen HY, LaBaer J, Tao N - Sci Rep (2014)

Bottom Line: Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy.We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding.This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.

ABSTRACT
Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

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Molecular-scale mechanism of ineffective Her2 population.A) Resistant clones (H6 and C5) overexpress MUC4 while sensitive clones (E8 and C11) do not. B) Dual-staining of Her2 and MUC4 in one single cell showing negative co-localization for Her2 and MUC4. Similar features were observed in multiple cells, but only a representative one was shown here. Scale bars, 50 μm.
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f5: Molecular-scale mechanism of ineffective Her2 population.A) Resistant clones (H6 and C5) overexpress MUC4 while sensitive clones (E8 and C11) do not. B) Dual-staining of Her2 and MUC4 in one single cell showing negative co-localization for Her2 and MUC4. Similar features were observed in multiple cells, but only a representative one was shown here. Scale bars, 50 μm.

Mentions: In order to examine the role of MUC4 in the ineffective population, the following experiments were carried out. 1) The expression levels of MUC4 of H6, C5, E8 and C11 were determined by immunofluorescence staining (Fig. 5A). The results show that the MUC4 expression levels in the Herceptin-resistant clones are indeed much higher than those in the Herceptin-sensitive clones (Supplementary Information Section 7). 2) The abundance levels of Her2 in all the clones were studied with immunofluorescence staining by using another anti-Her2 polyclonal antibody that recognizes different binding sites in Her2. The results confirm the finding from the SPR kinetic data that overall Her2 expression level does not significantly decrease in Herceptin-resistant clones (Supplementary Information Section 8). 3) Dual-staining of Her2 (using Herceptin as primary antibody) and MUC4 for a given Herceptin-resistant C5 clone was carried out (Fig. 5B and Supplementary Information Section 9), showing that the regions with more MUC4 tend to have less Herceptin bound to Her2, and vice versa. Such negative co-localization was observed in lots of the studied cells with varied extents, this particular cell shown in Fig. 5B was selected for best presentation. Note that the Her2 staining result in Fig. 5B (green) only represents the distribution of effective Her2 population rather than the total Her2 population, since those regions with ineffective Her2 population would lose all the Herceptin during the rinsing stage in the assay. The present study suggests that MUC4 interacts with some of the Her2 in the drug resistant cells, which weakens the binding of Herceptin to Her2. This is believed to be associated with the ineffective Her2 population observed in the SPR sensorgrams. The experimental data in the present work are consistent with the alternation hypothesis. Additional studies are needed to further elucidate the mechanism.


In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance.

Wang W, Yin L, Gonzalez-Malerva L, Wang S, Yu X, Eaton S, Zhang S, Chen HY, LaBaer J, Tao N - Sci Rep (2014)

Molecular-scale mechanism of ineffective Her2 population.A) Resistant clones (H6 and C5) overexpress MUC4 while sensitive clones (E8 and C11) do not. B) Dual-staining of Her2 and MUC4 in one single cell showing negative co-localization for Her2 and MUC4. Similar features were observed in multiple cells, but only a representative one was shown here. Scale bars, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196117&req=5

f5: Molecular-scale mechanism of ineffective Her2 population.A) Resistant clones (H6 and C5) overexpress MUC4 while sensitive clones (E8 and C11) do not. B) Dual-staining of Her2 and MUC4 in one single cell showing negative co-localization for Her2 and MUC4. Similar features were observed in multiple cells, but only a representative one was shown here. Scale bars, 50 μm.
Mentions: In order to examine the role of MUC4 in the ineffective population, the following experiments were carried out. 1) The expression levels of MUC4 of H6, C5, E8 and C11 were determined by immunofluorescence staining (Fig. 5A). The results show that the MUC4 expression levels in the Herceptin-resistant clones are indeed much higher than those in the Herceptin-sensitive clones (Supplementary Information Section 7). 2) The abundance levels of Her2 in all the clones were studied with immunofluorescence staining by using another anti-Her2 polyclonal antibody that recognizes different binding sites in Her2. The results confirm the finding from the SPR kinetic data that overall Her2 expression level does not significantly decrease in Herceptin-resistant clones (Supplementary Information Section 8). 3) Dual-staining of Her2 (using Herceptin as primary antibody) and MUC4 for a given Herceptin-resistant C5 clone was carried out (Fig. 5B and Supplementary Information Section 9), showing that the regions with more MUC4 tend to have less Herceptin bound to Her2, and vice versa. Such negative co-localization was observed in lots of the studied cells with varied extents, this particular cell shown in Fig. 5B was selected for best presentation. Note that the Her2 staining result in Fig. 5B (green) only represents the distribution of effective Her2 population rather than the total Her2 population, since those regions with ineffective Her2 population would lose all the Herceptin during the rinsing stage in the assay. The present study suggests that MUC4 interacts with some of the Her2 in the drug resistant cells, which weakens the binding of Herceptin to Her2. This is believed to be associated with the ineffective Her2 population observed in the SPR sensorgrams. The experimental data in the present work are consistent with the alternation hypothesis. Additional studies are needed to further elucidate the mechanism.

Bottom Line: Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy.We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding.This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.

ABSTRACT
Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

Show MeSH
Related in: MedlinePlus