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Mild hyperthermia influence on Herceptin(®) properties.

Escoffre JM, Deckers R, Sasaki N, Bos C, Moonen C - Radiol Oncol (2015)

Bottom Line: Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged.The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT.Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

View Article: PubMed Central - PubMed

Affiliation: Imaging Division, UMC Utrecht, Utrecht, the Netherlands.

ABSTRACT

Background: Mild hyperthermia (mHT) increases the tumor perfusion and vascular permeability, and reduces the interstitial fluid pressure, resulting in better intra-tumoral bioavailability of low molecular weight drugs. This approach is potentially also attractive for delivery of therapeutic macromolecules, such as antibodies. Here, we investigated the effects of mHT on the stability, immunological and pharmacological properties of Herceptin(®), a clinically approved antibody, targeting the human epidermal growth factor receptor 2 (HER-2) overexpressed in breast cancer.

Results: Herceptin(®) was heated to 37°C (control) and 42°C (mHT) for 1 hour. Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged. Immunological and pharmacological properties of Herceptin(®) were evaluated following mHT using HER-2 positive breast cancer cells (BT-474). Exposure of Herceptin(®) to mHT preserved recognition and binding affinity of Herceptin(®) to HER-2. Western-blot and cell proliferation assays on BT-474 cells showed that mHT left the inhibitory activities of Herceptin(®) unchanged.

Conclusions: The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT. Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

No MeSH data available.


Related in: MedlinePlus

Degradation and Dephosphorylation of HER-2 receptors by Herceptin®. 4×105 BT-474 cells were seeded in a 6-well plate and treated with Herceptin® (50 μg/mL) for 6 days. Immunoblots of protein lysates were analyzed for total and phosphorylated HER-2 receptors. Representative immunoblots of one experiment out of three independent experiments are shown. The number at the bottom of each lane indicates the relative fold change versus the control after normalization with the β-ACTIN signal. Data expressed as mean ± SD calculated from three independent experiments. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p < 0.05 (NS, non-significant; *p < 0.05 compared to the positive condition).
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f4-rado-49-01-41: Degradation and Dephosphorylation of HER-2 receptors by Herceptin®. 4×105 BT-474 cells were seeded in a 6-well plate and treated with Herceptin® (50 μg/mL) for 6 days. Immunoblots of protein lysates were analyzed for total and phosphorylated HER-2 receptors. Representative immunoblots of one experiment out of three independent experiments are shown. The number at the bottom of each lane indicates the relative fold change versus the control after normalization with the β-ACTIN signal. Data expressed as mean ± SD calculated from three independent experiments. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p < 0.05 (NS, non-significant; *p < 0.05 compared to the positive condition).

Mentions: To investigate how the binding of heated Herceptin® to HER-2 receptors affects them, their total and phosphorylated levels were monitored using western-blot assay. As expected, the exposure of BT-474 cells to native Herceptin® (50 μg/mL) resulted in significant decrease in relative densities of HER-2 receptors and phosphorylated HER-2 receptors compared to untreated cells (p < 0.05) (Figure 4). These results suggest that native Herceptin® leads to the internalization and the degradation of HER-2 receptors, thus preventing their phosphorylation in BT-474 cells. In addition, heated Herceptin® led to a decrease in total and phosphorylated HER-2 receptor levels that were comparable to the native Herceptin® (p > 0.05) (Figure 4).


Mild hyperthermia influence on Herceptin(®) properties.

Escoffre JM, Deckers R, Sasaki N, Bos C, Moonen C - Radiol Oncol (2015)

Degradation and Dephosphorylation of HER-2 receptors by Herceptin®. 4×105 BT-474 cells were seeded in a 6-well plate and treated with Herceptin® (50 μg/mL) for 6 days. Immunoblots of protein lysates were analyzed for total and phosphorylated HER-2 receptors. Representative immunoblots of one experiment out of three independent experiments are shown. The number at the bottom of each lane indicates the relative fold change versus the control after normalization with the β-ACTIN signal. Data expressed as mean ± SD calculated from three independent experiments. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p < 0.05 (NS, non-significant; *p < 0.05 compared to the positive condition).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362605&req=5

f4-rado-49-01-41: Degradation and Dephosphorylation of HER-2 receptors by Herceptin®. 4×105 BT-474 cells were seeded in a 6-well plate and treated with Herceptin® (50 μg/mL) for 6 days. Immunoblots of protein lysates were analyzed for total and phosphorylated HER-2 receptors. Representative immunoblots of one experiment out of three independent experiments are shown. The number at the bottom of each lane indicates the relative fold change versus the control after normalization with the β-ACTIN signal. Data expressed as mean ± SD calculated from three independent experiments. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p < 0.05 (NS, non-significant; *p < 0.05 compared to the positive condition).
Mentions: To investigate how the binding of heated Herceptin® to HER-2 receptors affects them, their total and phosphorylated levels were monitored using western-blot assay. As expected, the exposure of BT-474 cells to native Herceptin® (50 μg/mL) resulted in significant decrease in relative densities of HER-2 receptors and phosphorylated HER-2 receptors compared to untreated cells (p < 0.05) (Figure 4). These results suggest that native Herceptin® leads to the internalization and the degradation of HER-2 receptors, thus preventing their phosphorylation in BT-474 cells. In addition, heated Herceptin® led to a decrease in total and phosphorylated HER-2 receptor levels that were comparable to the native Herceptin® (p > 0.05) (Figure 4).

Bottom Line: Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged.The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT.Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

View Article: PubMed Central - PubMed

Affiliation: Imaging Division, UMC Utrecht, Utrecht, the Netherlands.

ABSTRACT

Background: Mild hyperthermia (mHT) increases the tumor perfusion and vascular permeability, and reduces the interstitial fluid pressure, resulting in better intra-tumoral bioavailability of low molecular weight drugs. This approach is potentially also attractive for delivery of therapeutic macromolecules, such as antibodies. Here, we investigated the effects of mHT on the stability, immunological and pharmacological properties of Herceptin(®), a clinically approved antibody, targeting the human epidermal growth factor receptor 2 (HER-2) overexpressed in breast cancer.

Results: Herceptin(®) was heated to 37°C (control) and 42°C (mHT) for 1 hour. Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged. Immunological and pharmacological properties of Herceptin(®) were evaluated following mHT using HER-2 positive breast cancer cells (BT-474). Exposure of Herceptin(®) to mHT preserved recognition and binding affinity of Herceptin(®) to HER-2. Western-blot and cell proliferation assays on BT-474 cells showed that mHT left the inhibitory activities of Herceptin(®) unchanged.

Conclusions: The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT. Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

No MeSH data available.


Related in: MedlinePlus