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Abstract
Colorectal cancer (CRC) is a disease with constantly increasing incidence and high mortality. The treatment efficacy could be curtailed by drug resistance resulting from poor drug penetration into tumor tissue and the tumor-specific microenvironment, such as hypoxia and acidosis. Furthermore, CRC tumors can be exposed to different pH depending on the position in the intestinal tract. CRC tumors often share upregulation of the Akt signaling pathway. In this study, we investigated the role of external pH in control of cytotoxicity of perifosine, the Akt signaling pathway inhibitor, to CRC cells using 2D and 3D tumor models. In 3D settings, we employed an innovative strategy for simultaneous detection of spatial drug distribution and biological markers of proliferation/apoptosis using a combination of mass spectrometry imaging and immunohistochemistry. In 3D conditions, low and heterogeneous penetration of perifosine into the inner parts of the spheroids was observed. The depth of penetration depended on the treatment duration but not on the external pH. However, pH alteration in the tumor microenvironment affected the distribution of proliferation- and apoptosis-specific markers in the perifosine-treated spheroid. Accurate co-registration of perifosine distribution and biological response in the same spheroid section revealed dynamic changes in apoptotic and proliferative markers occurring not only in the perifosine-exposed cells, but also in the perifosine-free regions. Cytotoxicity of perifosine to both 2D and 3D cultures decreased in an acidic environment below pH 6.7. External pH affects cytotoxicity of the other Akt inhibitor, MK-2206, in a similar way. Our innovative approach for accurate determination of drug efficiency in 3D tumor tissue revealed that cytotoxicity of Akt inhibitors to CRC cells is strongly dependent on pH of the tumor microenvironment. Therefore, the effect of pH should be considered during the design and pre-clinical/clinical testing of the Akt-targeted cancer therapy.
Highlights
Colorectal carcinoma (CRC) is the fourth most common cause of cancer death in Western countries [1, 2]
After 48 h, slight acidification of the sodium lactate (NaL)- and NaHCO3-supplemented medium was detected, presumably due to cellular metabolism (Figure 1A). These results verified that the cells in our cultures were exposed to physiological pH values within the range of those found either in the intestinal tract or in the extracellular space of tumors
We found that cytotoxicity of perifosine used in clinically relevant 10 and 20 μM concentrations [60, 61] for 24 h was suppressed by both acidosis alone and acidosis associated with hypoxia, as assessed by the MTT and adenosine triphosphate (ATP) assays (Figures 1B, C)
Summary
Colorectal carcinoma (CRC) is the fourth most common cause of cancer death in Western countries [1, 2]. Akt transduces signals regulating multiple biological processes, such as cell proliferation, survival, growth, angiogenesis, migration and epithelial-mesenchymal transition in CRC as well as other cancers [6, 7]. One of the most promising Akt inhibitors, perifosine, is a synthetic alkyl phospholipid that targets signal transduction pathways at the cell membrane by preventing correct localization of the Akt kinase, precluding its phosphorylation [9,10,11,12]. Perifosine was shown to effectively target the PI3K/Akt pathway both in preclinical models and in clinical trials [10, 11]. Clinical studies resulted in disappointing response rates of common solid tumors to perifosine as a single agent, while combination with another therapies seemed to be more effective [13,14,15]
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