The culture of 3D spheroids is a promising tool in drug development and testing. Recently, we synthesized a new group of compounds, unsymmetrical bisacridines (UAs), which exhibit high cytotoxicity against various human cell lines and antitumor potency against several xenografts. Here, we describe the ability of four UAs—C-2028, C-2041, C-2045, and C-2053—to influence the growth of HCT116 and H460 spheres and the viability of HCT116 cells in 3D culture compared with that in 2D standard monolayer culture. Spheroids were generated using ultra-low-attachment plates. The morphology and diameters of the obtained spheroids and those treated with UAs were observed and measured under the microscope. The viability of cells exposed to UAs at different concentrations and for different incubation times in 2D and 3D cultures was assessed using 7-AAD staining. All UAs managed to significantly inhibit the growth of HCT116 and H460 spheroids. C-2045 and C-2053 caused the death of the largest population of HCT116 spheroid cells. Although C-2041 seemed to be the most effective in the 2D monolayer experiments, in 3D conditions, it turned out to be the weakest compound. The 3D spheroid culture seems to be a suitable method to examine the efficiency of new antitumor compounds, such as unsymmetrical bisacridines.
There is increasing evidence that the expression level of drug metabolic enzymes affects the final cellular response following drug treatment. Moreover, anti-tumour agents may modulate enzymatic activity and/or cellular expression of metabolic enzymes in tumour cells. We have investigated the influence of CYP3A4 overexpression on the cellular response induced by the anti-tumour agent C-1311 in hepatoma cells. C-1311-mediated CYP3A4 activity modulation and the effect of CYP3A4 overexpression on C-1311 metabolism have also been examined. With the HepG2 cell line and its CYP3A4-overexpressing variant, Hep3A4, experiments involving DAPI staining, cell cycle analysis, phosphatidylserine externalisation and senescence-associated (SA)-β-galactosidase expression, were used to monitor the effects of C-1311 exposure. C-1311 cellular metabolism and CYP3A4 activity were investigated by high-performance liquid chromatography. C-1311 metabolism was very low in both hepatoma cell lines and slightly influenced by CYP3A4 expression. Interestingly, in HepG2 cells, C-1311 was an effective modulator of CYP3A4 enzymatic activity, being the inhibitor of this isoenzyme in Hep3A4 cells. Cell cycle analysis showed that HepG2 cells underwent a rather stable G(2) /M arrest following C-1311 exposure, whereas CYP3A4-overexpressing cells accumulated only slightly in this compartment. C-1311-treated cells died by apoptosis and necrosis, whereas surviving cells underwent senescence; however, these effects occurred faster and more intensely in Hep3A4 cells. Although CYP3A4 did not influence C-1311 metabolism, changes in CYP3A4 levels affected the C-1311-induced response in hepatoma cells. Therefore, inter-patient differences in CYP3A4 levels should be considered when assessing the potential therapeutic effects of C-1311.
The acridinone derivates 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) are promising antitumor agents with high activity against several experimental cellular and tumor models and are under evaluation in preclinical and early phase clinical trials. Recent evidence from our laboratories has indicated that both compounds were conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. The present studies were designed to test the ability and selectivity of UGT1A10 in the glucuronidation of acridinone antitumor agents in a cellular context. We show that in KB-3 cells, a HeLa subline lacking expression of any UGT isoforms, both C-1305 and C-1311 undergo metabolic transformation to the glucuronidated forms on overexpression of UGT1A10. Furthermore, UGT1A10 overexpression significantly increased the cytotoxicity of C-1305, but not C-1311, suggesting that the glucuronide was more potent than the C-1305 parent compound. These responses were selective for UGT1A10 because documented overexpression of UGT2B4 failed to produce glucuronide products and failed to alter the cytotoxicity for both compounds. These findings contribute to our understanding of the mechanisms of action of these agents and are of particular significance because data for C-1305 contradict the dogma that glucuronidation typically plays a role in detoxification or deactivation. In summary, these studies suggest that extrahepatic UGT1A10 plays an important role in the metabolism and the bioactivation of C-1305 and constitutes the basis for further mechanistic studies on the mode of action of this drug, as well as translational studies on the role of this enzyme in regulation of C-1305 toxicity in cancer.
Aim: To examine whether CYP3A4 overexpression influences the metabolism of anticancer agent imidazoacridinone C-1311 in CHO cells and the responses of the cells to C-1311. Methods: Wild type CHO cells (CHO-WT), CHO cells overexpressing cytochrome P450 reductase (CPR) [CHO-HR] and CHO cells coexpressing CPR and CYP3A4 (CHO-HR-3A4) were used. Metabolic transformation of C-1311 and CYP3A4 activity were measured using RP-HPLC. Flow cytometry analyses were used to examine cell cycle, caspase-3 activity and cell apoptosis. The expression of pH 6.0-dependent β-galactosidase (SA-β-gal) was studied to evaluate accelerated senescence. ROS generation was analyzed with CM-H 2 DCFDA staining. Results: CYP3A4 overexpression did not change the metabolism of C-1311 in CHO cells: the levels of all metabolites of C-1311 increased with the exposure time to a similar extent, and the differences in the peak level of the main metabolite M3 were statistically insignificant among the three CHO cell lines. In CHO-HR-3A4 cells, C-1311 effectively inhibited CYP3A4 activity without affecting CYP3A4 protein level. In the presence of C-1311, CHO-WT cells underwent rather stable G 2 /M arrest, while the two types of transfected cells only transiently accumulated at this phase. C-1311-induced apoptosis and necrosis in the two types of transfected cells occurred with a significantly faster speed and to a greater extent than in CHO-WT cells. Additionally, C-1311 induced ROS generation in the two types of transfected cells, but not in CHO-WT cells. Moreover, CHO-HR-3A4 cells that did not die underwent accelerated senescence. Conclusion: CYP3A4 overexpression in CHO cells enhances apoptosis induced by C-1311, whereas the metabolism of C-1311 is minimal and does not depend on CYP3A4 expression.
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