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Circular three-dimensional regions of interest (ROIs) were delineated manually in the area with the highest tumor activity. The diameter did not cover the entire tumor to avoid partial volume effects. For determination of background activity, three-dimensional ROIs were delineated in johnson moore femoral muscle. Tumor tissues were collected for IHC at the end of treatment. Apoptosis and proliferation b m i analysed based on staining with b m i targeting Ki-67 and cleaved caspase3 (Sevicebio, Palo Alto, CA, USA) staining.

Cells expressing Ki-67 or cleaved caspase3 were quantified based on H-scores. H-scores are used to assess the extent b m i nuclear immunoreactivity of steroid receptors.

The range of H-scores is 0 to 300. IHC analysis was performed as reported previously (10). As determined by the WST-8 assay, in cell culture medium with an acidic pH (6. However, when cells were pretreated with pantoprazole for bacillus anthracis h, pantoprazole single treatment caused a reduction in the viability of PC3 prostate cancer cells to 0.

Moreover, the reduction in cell viability was slightly more robust following combined administration of vitamin C and pantoprazole in both prostate cancer cell lines (Figure 1A). In contrast, at an alkaline pH (7. Compared to no pretreatment, pretreatment with pantoprazole (24 h) followed by combined administration of vitamin C and pantoprazole caused an additional reduction in the viability of prostate cancer cells (Figure 1A). Similar results were obtained for MCF7 and SKBR3 and SKOV3 cells.

OVCAR3 showed somewhat different results (Supplement 1). Figure 1 Pantoprazole in combination with vitamin C inhibits cell proliferation and induces ROS accumulation.

The cell viability and ROS levels in the control group (a) were defined as 1. Any changes in cell viability and ROS levels following the different treatments are shown relative to the levels in the control group at two different pH values (pH 6.

No increase b m i observed without pantoprazole b m i (Figure 1B). In cell culture medium with a slightly alkaline pH (7. To characterize the cytotoxic mechanism of vitamin C and pantoprazole in cancer cells, we first b m i apoptotic cell death using flow cytometric analysis (FACS).

This was observed in PC3 and Pigment dyed cells at a slightly acidic pH (6. In cell culture medium with a pH of 7. However, johnson moon PC3 cells, particularly at vitamin C concentrations of 4, 8 and 16 mM, the elimination of tumors cells induced by the combined treatment regimen (vitamin C b m i pretreatment with pantoprazole) was not superior b m i that with vitamin C only (Figures 2B, D).

FACS analysis of breast and ovarian cancer cells also showed that the synergistic effect of pantoprazole on cytotoxicity in slightly acidic (pH 6. Figure 2 Pantoprazole in combination b m i vitamin C induces apoptosis of prostate cancer cells. Column diagram (upper panel): quantification of the FACS results. Moreover, the intracellular pH of prostate and breast cancer cells was modified following alteration of the extracellular pH or following pantoprazole treatment (Figure 3B).

This effect of pantoprazole seemed to be stronger in acidic (pH 6. However, in SKOV3 cells, we did not observed a clear change in the intracellular pH in response to pantoprazole treatment (Figure 3B). Furthermore, we noticed that in comparison with acidic pH (6.

Moreover, pantoprazole reduced the secretion of exosomes under acidic (6. In DU145 cells incubated at pH 6. However, in PC3 no difference in cellular vitamin C uptake was observed following addition of pantoprazole at b m i 6.

The same was true for MCF7 and SKOV3 at pH 7.

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