Limits...
Photochemical activation of MH3-B1/rGel: a HER2-targeted treatment approach for ovarian cancer.

Bull-Hansen B, Berstad MB, Berg K, Cao Y, Skarpen E, Fremstedal AS, Rosenblum MG, Peng Q, Weyergang A - Oncotarget (2015)

Bottom Line: Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells.The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts.Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

ABSTRACT
HER2-targeted therapy has been shown to have limited efficacy in ovarian cancer despite frequent overexpression of this receptor. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, currently undergoing clinical evaluation. In the present project we studied the application of PCI in combination with the HER2-targeted recombinant fusion toxin, MH3-B1/rGel, for the treatment of ovarian cancer. The SKOV-3 cell line, resistant to trastuzumab- and MH3-B1/rGel- monotherapy, was shown to respond strongly to PCI of MH3-B1/rGel to a similar extent as observed for the treatment-sensitive SK-BR-3 breast cancer cells. Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells. This was shown by the positive Pearson's correlation coefficient between Alexa488-labeled MH3-B1/rGel and Lysotracker in SKOV-3 cells in contrast to the negative Pearson's correlation coefficient in SK-BR-3 cells. The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts. Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines. The presented results warrant future development of PCI in combination with MH3-B1/rGel as a novel therapeutic approach in preclinical models of ovarian cancer.

No MeSH data available.


Related in: MedlinePlus

Cytotoxicity, uptake and cellular localization of MH3-B1/rGel after 4 hrs incubationRelative cell viability (MTT) of SK-BR-3 (A) and SKOV-3 (B) following 4 hrs incubation with MH3-B1/rGel, rGel and MH3-B1, representative sigmoidal curves of three experiments, (fit model: a/(1+exp(−(x-x0)/b))), error bars = SD. (C): Cellular uptake of 2 nM Alexa488-MH3-B1/rGel following 4 hrs incubation in SK-BR-3 and SKOV-3 cells, representative flow cytometry charts of three experiments. (D): Total cellular Alexa488-MH3-B1/rGel fluorescence, average of three experiments, error bars = SE. (E): SK-BR-3 and SKOV-3 cells following 4 hrs incubation of 2 nM Alexa-488-MH3-B1/rGel. Alexa488-MH3-B1/rGel (green), LTR (blue) and colocalization of the two (white). The confocal micrographs are representative of three independent experiments. The colocalization of Alexa488-MH3-B1/rGel with LTR was calculated by the Manders' thresholded coefficient from the total cellular accumulation measured (F) and the Pearson's correlation coefficient was used to calculate the correlation between Alexa488-MH3-B1/rGel and LTR based on fluorescence intensities/pixel (G), F and G shows average of three experiments, error bars = SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494949&req=5

Figure 2: Cytotoxicity, uptake and cellular localization of MH3-B1/rGel after 4 hrs incubationRelative cell viability (MTT) of SK-BR-3 (A) and SKOV-3 (B) following 4 hrs incubation with MH3-B1/rGel, rGel and MH3-B1, representative sigmoidal curves of three experiments, (fit model: a/(1+exp(−(x-x0)/b))), error bars = SD. (C): Cellular uptake of 2 nM Alexa488-MH3-B1/rGel following 4 hrs incubation in SK-BR-3 and SKOV-3 cells, representative flow cytometry charts of three experiments. (D): Total cellular Alexa488-MH3-B1/rGel fluorescence, average of three experiments, error bars = SE. (E): SK-BR-3 and SKOV-3 cells following 4 hrs incubation of 2 nM Alexa-488-MH3-B1/rGel. Alexa488-MH3-B1/rGel (green), LTR (blue) and colocalization of the two (white). The confocal micrographs are representative of three independent experiments. The colocalization of Alexa488-MH3-B1/rGel with LTR was calculated by the Manders' thresholded coefficient from the total cellular accumulation measured (F) and the Pearson's correlation coefficient was used to calculate the correlation between Alexa488-MH3-B1/rGel and LTR based on fluorescence intensities/pixel (G), F and G shows average of three experiments, error bars = SE.

Mentions: PCI is a method for cytosolic release of drugs entrapped in endosomes and lysosomes. PCI in vitro is usually performed by a 1-18 hrs incubation of the macromolecule of interest prior to light exposure [32, 17]. This relatively short incubation time of the macromolecule is utilized to avoid strong influence of lysosomal degradation on the PCI outcome. In the present in vitro protocol, a 4 hrs pulse of 2 nM MH3-B1/rGel was used prior to the light exposure. Since the intracellular trafficking of MH3-B1/rGel in the post-treatment period is expected to influence on the therapeutic effect, a cytotoxicity evaluation was performed 72 hrs after the 4 hrs pulse of MH3-B1/rGel to verify that SKOV-3 cells were resistant to MH3-B1/rGel also after a 4 hrs treatment (Fig. 2A and 2B). The TI's after 4 hrs treatment were found to be higher in both cell lines compared to that after 72 hrs treatment. However, the SKOV-3 cells were found to be relatively less sensitive to the treatment as e.g. 2 nM MH3-B1/rGel reduced cell viability by ~35 % in SK-BR-3 cells (Fig. 2B), but did not induce detectable cytotoxicity in the SKOV-3 cell line (Fig. 2A). Calculating the TI at IC35 of MH3-B1/rGel vs. rGel (TI35) revealed an ~11-fold higher TI35 in SK-BR-3 cells compared to SKOV-3 cells after 4 hrs treatment with MH3-B1/rGel. SKOV-3 cells were therefore found resistant to MH3-B1/rGel also when administrated for 4 hrs compared to SK-BR-3 cells. Four hrs incubation of MH3-B1 (without rGel) did not induce any significant cytotoxicity in any of the cell lines, indicating MH3-B1/rGel-mediated toxicity to be generated through ribosomal inhibition (Fig. 2A and 2B).


Photochemical activation of MH3-B1/rGel: a HER2-targeted treatment approach for ovarian cancer.

Bull-Hansen B, Berstad MB, Berg K, Cao Y, Skarpen E, Fremstedal AS, Rosenblum MG, Peng Q, Weyergang A - Oncotarget (2015)

Cytotoxicity, uptake and cellular localization of MH3-B1/rGel after 4 hrs incubationRelative cell viability (MTT) of SK-BR-3 (A) and SKOV-3 (B) following 4 hrs incubation with MH3-B1/rGel, rGel and MH3-B1, representative sigmoidal curves of three experiments, (fit model: a/(1+exp(−(x-x0)/b))), error bars = SD. (C): Cellular uptake of 2 nM Alexa488-MH3-B1/rGel following 4 hrs incubation in SK-BR-3 and SKOV-3 cells, representative flow cytometry charts of three experiments. (D): Total cellular Alexa488-MH3-B1/rGel fluorescence, average of three experiments, error bars = SE. (E): SK-BR-3 and SKOV-3 cells following 4 hrs incubation of 2 nM Alexa-488-MH3-B1/rGel. Alexa488-MH3-B1/rGel (green), LTR (blue) and colocalization of the two (white). The confocal micrographs are representative of three independent experiments. The colocalization of Alexa488-MH3-B1/rGel with LTR was calculated by the Manders' thresholded coefficient from the total cellular accumulation measured (F) and the Pearson's correlation coefficient was used to calculate the correlation between Alexa488-MH3-B1/rGel and LTR based on fluorescence intensities/pixel (G), F and G shows average of three experiments, error bars = SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494949&req=5

Figure 2: Cytotoxicity, uptake and cellular localization of MH3-B1/rGel after 4 hrs incubationRelative cell viability (MTT) of SK-BR-3 (A) and SKOV-3 (B) following 4 hrs incubation with MH3-B1/rGel, rGel and MH3-B1, representative sigmoidal curves of three experiments, (fit model: a/(1+exp(−(x-x0)/b))), error bars = SD. (C): Cellular uptake of 2 nM Alexa488-MH3-B1/rGel following 4 hrs incubation in SK-BR-3 and SKOV-3 cells, representative flow cytometry charts of three experiments. (D): Total cellular Alexa488-MH3-B1/rGel fluorescence, average of three experiments, error bars = SE. (E): SK-BR-3 and SKOV-3 cells following 4 hrs incubation of 2 nM Alexa-488-MH3-B1/rGel. Alexa488-MH3-B1/rGel (green), LTR (blue) and colocalization of the two (white). The confocal micrographs are representative of three independent experiments. The colocalization of Alexa488-MH3-B1/rGel with LTR was calculated by the Manders' thresholded coefficient from the total cellular accumulation measured (F) and the Pearson's correlation coefficient was used to calculate the correlation between Alexa488-MH3-B1/rGel and LTR based on fluorescence intensities/pixel (G), F and G shows average of three experiments, error bars = SE.
Mentions: PCI is a method for cytosolic release of drugs entrapped in endosomes and lysosomes. PCI in vitro is usually performed by a 1-18 hrs incubation of the macromolecule of interest prior to light exposure [32, 17]. This relatively short incubation time of the macromolecule is utilized to avoid strong influence of lysosomal degradation on the PCI outcome. In the present in vitro protocol, a 4 hrs pulse of 2 nM MH3-B1/rGel was used prior to the light exposure. Since the intracellular trafficking of MH3-B1/rGel in the post-treatment period is expected to influence on the therapeutic effect, a cytotoxicity evaluation was performed 72 hrs after the 4 hrs pulse of MH3-B1/rGel to verify that SKOV-3 cells were resistant to MH3-B1/rGel also after a 4 hrs treatment (Fig. 2A and 2B). The TI's after 4 hrs treatment were found to be higher in both cell lines compared to that after 72 hrs treatment. However, the SKOV-3 cells were found to be relatively less sensitive to the treatment as e.g. 2 nM MH3-B1/rGel reduced cell viability by ~35 % in SK-BR-3 cells (Fig. 2B), but did not induce detectable cytotoxicity in the SKOV-3 cell line (Fig. 2A). Calculating the TI at IC35 of MH3-B1/rGel vs. rGel (TI35) revealed an ~11-fold higher TI35 in SK-BR-3 cells compared to SKOV-3 cells after 4 hrs treatment with MH3-B1/rGel. SKOV-3 cells were therefore found resistant to MH3-B1/rGel also when administrated for 4 hrs compared to SK-BR-3 cells. Four hrs incubation of MH3-B1 (without rGel) did not induce any significant cytotoxicity in any of the cell lines, indicating MH3-B1/rGel-mediated toxicity to be generated through ribosomal inhibition (Fig. 2A and 2B).

Bottom Line: Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells.The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts.Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

ABSTRACT
HER2-targeted therapy has been shown to have limited efficacy in ovarian cancer despite frequent overexpression of this receptor. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, currently undergoing clinical evaluation. In the present project we studied the application of PCI in combination with the HER2-targeted recombinant fusion toxin, MH3-B1/rGel, for the treatment of ovarian cancer. The SKOV-3 cell line, resistant to trastuzumab- and MH3-B1/rGel- monotherapy, was shown to respond strongly to PCI of MH3-B1/rGel to a similar extent as observed for the treatment-sensitive SK-BR-3 breast cancer cells. Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells. This was shown by the positive Pearson's correlation coefficient between Alexa488-labeled MH3-B1/rGel and Lysotracker in SKOV-3 cells in contrast to the negative Pearson's correlation coefficient in SK-BR-3 cells. The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts. Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines. The presented results warrant future development of PCI in combination with MH3-B1/rGel as a novel therapeutic approach in preclinical models of ovarian cancer.

No MeSH data available.


Related in: MedlinePlus