Hypoglycemic/hypoxic condition in vitro mimicking the tumor microenvironment markedly reduced the efficacy of anticancer drugs.
ABSTRACT Tumor tissues are often hypoxic because of defective vasculature. We previously showed that tumor tissues are also often deprived of glucose. The efficacy of anticancer drugs is affected by the tumor microenvironment, partly because of the drug delivery and cellular drug resistance; however, the precise mechanisms remain to be clarified. In the present study, we attempted to clarify whether hypoglycemic/hypoxic condition, which mimics the tumor microenvironment, might induce drug resistance, and if it did, to elucidate the underlying mechanisms. Pancreatic cancer-derived PANC-1 cells were treated with serial dilutions of anticancer drugs and incubated in either normoglycemic (1.0 g/L glucose) or hypoglycemic (0 g/L glucose) and normoxic (21% O(2)) or hypoxic (1% O(2) ) conditions. The 50% inhibitory concentration of gemcitabine was 1000 times higher for PANC-1 cells incubated under the hypoglycemic/hypoxic condition than for those incubated under the normoglycemic/normoxic condition. Conventional anticancer drugs target rapidly growing cells, so that non-proliferating or slowly proliferating cells usually show resistance to drugs. Though the cell cycle was delayed, sufficient cellular uptake and DNA incorporation of gemcitabine occurred under the hypoglycemic/hypoxic condition to cause DNA lesions and S-phase arrest. To overcome hypoglycemic/hypoxia-induced drug resistance, we examined kinase inhibitors targeting Chk1 or cell-survival signaling pathways. Among the compounds examined, the combination of UCN-01 and LY294002 partially sensitized the cells to gemcitabine under the hypoglycemic/hypoxic condition. These findings suggested that the adoption of suitable strategies may enhance the cytotoxicities of clinically used anticancer drugs against cancer cells.
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ABSTRACT: In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures' response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli.PLoS ONE 01/2012; 7(9):e44640. · 3.73 Impact Factor
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ABSTRACT: Hypoxia is part of the tumor microenvironment favoring cancer resistance to chemotherapy mediated by mutations in the tumor suppressor p53 gene (TP53), or by conformational wt TP53 dysfunction. Since it is important to suppress tumor adaptation to hypoxia, irrespective of p53 status, we compared the efficacy of nutlin-3 which prevents MDM2-wt p53 interactions and PRIMA-1 which promotes mutant p53 reactivation and induction of massive apoptosis, under normoxia and hypoxia, against (a) SKBR3 breast carcinoma harboring a mutant p53R175H and over-expressing erbB2; and (b) genetically matched breast cancer ERα positive MCF-7 cells harboring either wt p53 or mutant p53 R175H. Under normoxia, PRIMA-1 was active against breast cancer cells harboring mutant p53. However, hypoxia further increased the susceptibility of mutant p53 breast cancer SKBR3 cells to lower PRIMA-1 levels, possibly through oxidative stress since this was counteracted by N-acetylcysteine. When using MCF-7 cells over-expressing mutant p53, PRIMA-1 synergized with exogenous peroxidase to increase apoptosis concomitantly with induction of PUMA and Mn-SOD, under normoxia. Wt p53 MCF-7 cells responded to hypoxia by increasing superoxide dismutase and their reactivity with the PAb240 antibody, known to recognize conformationally-inactive p53. This correlated with sensitization of wt p53 MCF-7 cells to PRIMA-1 but not to nutlin-3. PRIMA-1 toxicity against normoxic wt p53 MCF-7 cells was also decreased by Mn-SOD over-expression or when added with the glutathione antagonist, buthionine sulfoximine. This report shows for the first time that hypoxia increases PRIMA-1 toxicity in human breast cancer cells, partly by modulating p53 conformation and by inducing superoxide turnover. Our results suggest that PRIMA-1 may help to prevent hypoxia-mediated tumor chemoresistance.Biochemical pharmacology 12/2012; 84(12):1563–1570. · 4.25 Impact Factor