Abstract
BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and is associated with substantial mortality. Because HCCs have strong resistance to conventional chemotherapeutic agents, novel therapeutic strategies are needed to improve survival in HCC patients.MethodsHere, we developed a fluorescence image-based phenotypic screening system in vitro to identify HCC-specific drugs in co-cultures of HCC cells with hepatocytes. To this end, we identified two distinctive markers of HCC, CHALV1 and AFP, which are highly expressed in HCC cell lines and liver cancer patient-derived materials. We applied these markers to an HCC-specific drug screening system.ResultsThrough pilot screening, we identified three anti-folate compounds that had HCC-specific cytotoxicity. Among them, pyrimethamine exhibited the greatest HCC-specific cytotoxicity. Interestingly, pyrimethamine significantly increased the size and number of lysosomes and subsequently induced the release of cathepsin B from the lysosome to the cytosol, which triggered caspase-3-dependent apoptosis in Huh7 (HCC) but not Fa2N-4 cells (immortalized hepatocytes). Importantly, Fa2N-4 cells had strong resistance to pyrimethamine relative to Huh7 cells in 2D and 3D culture systems.ConclusionThese results demonstrate that this in vitro image-based phenotypic screening platform has the potential to be widely adopted in drug discovery research, since we promptly estimated anticancer activity and hepatotoxicity and elucidated functional roles of pyrimethamine during the apoptosis process in HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2816-x) contains supplementary material, which is available to authorized users.
Highlights
Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and is associated with substantial mortality
CHALV1 and AFP are appropriate markers to distinguish between HCCs and hepatocytes We aimed to develop liver cancer-specific compounds that induce cell death in HCC cells, while minimizing the damage to hepatocytes, by creating a mixed cell culture system
Because two different populations were present in the co-culture system, we first sought to find HCC-specific markers to distinguish between the HCC cells and hepatocytes
Summary
Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and is associated with substantial mortality. We aimed to develop liver cancer-specific drugs that could induce cell death in HCC cells, while minimizing the damage to normal hepatocytes, in a mixed cell culture system containing hepatocytes and HCC cells. Since the inhibition of DHFR blocks nucleotide biosynthesis, anti-folate drugs reduce the proliferation of cancer cells [7]. Pyrimethamine (2,4diamino-5-p-chlorophenyl-6-ethyl-pyrimidine), a folic acid antagonist, is used to treat protozoal infections It is used as an antimalarial drug and a treatment for Toxoplasma gondii infections in immunocompromised patients [8,9,10]. We identified a hitherto unknown mechanism of pyrimethamine-induced apoptosis in HCC cells using fluorescence image-based phenotypic analysis. In order to assess pyrimethamineinduced phenotypic changes and cytotoxic effects in HCC, we applied various cell-based assay models in vitro to the High Content Screening system. We applied a hepatocellular 3D culture method to this system, which is the appropriate culture model to maintain liver-specific functions and to validate drug efficiency
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