Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia.
A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA).
Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs.
A panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia.
Solid tumors contain regions with mild (hypoxia) to severe oxygen deficiency (anoxia), due to the lack of blood supply to the growing tumor nodules [1–3]. Oxygen and nutrients are essential for solid tumor growth, and when sufficient oxygen is not provided growth arrest or necrosis occurs in the unvascularized tumor core [4, 5]. Neovascularization, or angiogenesis, is required to keep the growing tumor oxygenated and increased vascular density is correlated with increased metastasis and decreased patient survival in many cancers (reviewed by [6, 7]).
Decreased oxygenation leads to various biochemical responses in the tumor cells that ultimately can result in either adaptation or cell death. Hypoxia-inducible factor α (HIF-1α) is one of the most important transcription factors and a regulator of gene products during hypoxia . Initial or moderate increase of HIF-1α levels could lead to cell adaptation, and in the absence of oxygen cancer cells adjust to their new microenvironment mainly by angiogenesis stimulation by vascular endothelial growth factor (VEGF) , inhibition of apoptosis via Bcl-2 , modifying the cellular glucose/energy metabolism , adapting to acidic extracellular pH  and up-regulation of proteins involved in metastasis . The delicate balance between activators and inhibitors regulate adaptation or cell death in growing tumor nodules.
Hypoxia mediated resistance to radiotherapy and chemotherapy
Hypoxic cells may be resistant to both radiotherapy and conventional chemotherapy. Studies show that hypoxia has a negative impact of radiotherapy on tumor cells in various cancers such as mammary carcinoma , head and neck carcinoma  and uterine cervix carcinoma . There are several non-excluding theories to explain the fact that also conventional chemotherapy has less effect on hypoxic tumor cells. The anarchic vascular pattern characteristic of many tumors includes caliber changes, loops and trifurcations . This, and the distance between cell and blood vessel diminish the exposure of the anticancer drug and also the proliferation of the cells [4, 18]. Since the cytotoxic effect is greater in rapidly dividing cells, the slow proliferating tumor cells far away from the blood vessels is less sensitive to chemotherapy [1, 18]. Hypoxia also selects for cells with low expression of p53 and consequently p53-induced apoptosis is reduced in hypoxic cells . In normoxic surroundings DNA injuries caused by some anticancer drugs is more permanent, while in hypoxic surroundings higher levels of restoration occurs . Another association between hypoxia and chemotherapy resistance is the up-regulation of the multidrug resistance (MDR) genes and over expression of the gene product P-glycoprotein (P-gp), which is known to be involved in multidrug resistance [21, 22].
Different methods have been applied to study the effect of a cytotoxic drug in an environment resembling that of a tumor, i.e. with tumor cells in a hypoxic environment. However, earlier in vitro studies on drug effects in hypoxic cells have been performed with different methods and have also yielded different results. For example, hypoxic or anoxic cells may be generated by incubation of monolayer cultures in hypoxic incubators with constant O2, N2 and CO2 concentrations [23–26], or by use of airtight containers, in which the oxygen concentration in the gas phase is held at a constant level, incubated in aerobic incubators . The redox-potential in the medium can also be altered with, for example, cobalt chloride (CoCl2) to achieve chemical hypoxia  or enzyme generated oxygen depletion by adding glucose oxidase and catalase . A three-dimensional way of studying the effect of drugs in hypoxia is the use of tumor spheroids [30, 31]. Spheroids are generated by culturing adherent cells and give a 3D cellular context in which oxygen-, glucose- and ATP gradient varies . After treatment, cell survival is measured to determine the relative hypoxic toxicity of a drug. This has previously been done by for example clonogenic  or non-clonogenic colorimetric assays using MTT [23, 34, 35], sulforhodamine B  or by trypan blue staining [24, 26]. However, most of these investigations have been done with limited series of drugs and/or cell types, and slightly different conditions. In this work we have screened a larger panel of drugs in five different cell lines, to investigate their sensitivity to a panel of chemotherapeutic agents under conditions of normoxia (20% O2), hypoxia (1% O2), and anoxia (0.1% O2).
The in vitro analysis were carried out in a panel of cancer cell lines, including A2780 (ECACC Salisbury, UK), ACHN, MCF-7, NCI-H69 (all American Type Culture Collection, LGC Standards, Borås, Sweden) and U937-GTB (kind gift from Kennet Nilsson, Department of pathology, Uppsala University). The different cell lines were selected as representatives of various kinds of cancer types, including ovarian cancer (A2780), breast cancer (MCF-7), renal adenocarcinoma (ACHN), small cell lung cancer (H69) and a leukemic monocyte lymphoma (U937). Cell growth medium RPMI 1640 (Sigma-Aldrich, Stockholm, Sweden), supplemented with 10% heat-inactivated fetal bovine serum (FCS; Sigma-Aldrich, Stockholm, Sweden), 2 mmol/L L-glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin (Sigma-Aldrich, Stockholm, Sweden), was used to maintain A2780-, ACHN-, H69- and U937 cell lines. MCF-7 was maintained in Minimum Essential Medium Eagle (M5650, Sigma-Aldrich, Stockholm, Sweden), supplemented with 10% heat-inactivated FCS (Sigma-Aldrich, Stockholm, Sweden), 2 mmol/L L-glutamine, 100 μg/mL streptomycin, 100 U/mL penicillin (Sigma-Aldrich, Stockholm, Sweden) and 1 mM sodium pyruvate (P5280, Sigma-Aldrich, Stockholm, Sweden). All cell lines were kept in 75 cm2 culture flasks (TPP, Trasadingen, Switzerland) at 37°C in a humidified atmosphere of 95% air, 5% CO2. The enzyme accutase (PAA, Pasching, Austria) was used to detach the A2780-, ACHN- and HT29 cells from the bottom of the flask and accumax (PAA, Pasching, Austria) was used to separate the H69 cells and detach the MCF-7 cells from the flask.
Drugs and reagents
The drugs tested were selected as representatives of various chemotherapeutic drug groups with different modes of action. 5-fluorouracil (5-FU), cisplatin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan and vincristine were obtained from the Swedish Pharmacy (Uppsala Sweden). Acriflavine, digitoxin, digoxin, rapamycin, thalidomide and topotecan where purchased from Sigma-Aldrich (Stockholm, Sweden), mitomycin c from Medac (Varberg, Sweden), bortezomib and sorafenib from LC laboratories (Woburn, MA, USA) and tirapazamine from Chemos GmbH (Regenstauf, Germany). The drugs are listed in Table 1, including earlier reports of effect(s) in hypoxia. The pharmaceutical preparations were dissolved according to instructions from the manufacturer, the other drugs were dissolved in dimetylsulfoxid (DMSO; Sigma-Aldrich, Stockholm, Sweden) or dimethylacetamide (DMA; Sigma-Aldrich, Stockholm, Sweden) and stored frozen in −70°C for maximum three months. Sterile phosphate buffered saline (PBS; Sigma-Aldrich, Stockholm, Sweden) was used to dilute the drugs to desirable concentrations. Fluoresceindiacetate (FDA; Sigma-Aldrich, Stockholm, Sweden) was dissolved in DMSO to a concentration of 10 mg/mL and kept frozen (−20°C) as a stock solution protected from light.
The cells were seeded in duplicate in 96-well microtiter plates (NUNC, Roskilde, Denmark). 180 μL cell suspension, with the concentration of 100 000 cells/mL was added to each well, blank wells containing medium only. The normoxic set of plates was placed in an aerobic incubator (atmospheric) and the hypoxic/anoxic set where moved to a Ruskinn InVivo2 500 hypoxic incubator (Ruskinn Technology Ltd, Pencoed, UK) and where equilibrated at 37°C in a humidified atmosphere of 5% CO2 and limited oxygen, either 0.1% O2 or 1.0% O2. Hereafter 0.1% O2 is considered as extreme deprivation of oxygen and will be referred to as anoxia and 1.0% O2 will be referred to as hypoxia. After 18 hours pre-incubation, 20 μL of test solution were added to each well (PBS to blank and control, drug solution to duplicate test wells) and left to incubate for 72 hours. After the incubation, measurement according to the fluorometric microculture cytotoxicity assay (FMCA) was performed.
The Fluorometric Microculture Cytotoxicity Assay FMCA
The non-clonogenic cell viability assay FMCA is based on the fluorescence generated from the hydrolysis of fluoresceindiacetate (FDA) to fluorescein by cells with intact cell membranes. The methodology is described by Larsson et al. (1992) and also in detail in the protocol article by Lindhagen et al. (2008) [67, 68]. In short, cells (20000/well) were pre-incubated at normoxia, hypoxia or anoxia, where after drugs were added and the plates incubated for 72 hrs, washed ones with PBS in a microtiter plate washer (Multiwash, Dynatech Laboratories) and thereafter FDA (100 μl of 0.01 mg/mL FDA, Sigma-Aldrich, Stockholm, Sweden) in a buffer, was added. After 40 minutes incubation (37°C) the generated fluorescence was measured at 485/520 nm in a Fluoroskan II (Labsystems, Helsinki Oy, Finland) and the survival index (SI%) for each drug concentration was calculated. All experiments were performed three times. From the mean SI%-curves the half maximal inhibitory concentration (IC50) was determined using non-linear regression analysis in Prism 5 Software Package (Graph Pad, San Diego, CA). Cytotoxicity ratios (Ranox = anoxic IC50/normoxic IC50 and Rhypox = hypoxic IC50/normoxic IC50) were determined for each drug and cell line.
For the three obtained SI% replicates, Grubbs test was used to detect and exclude significant outliers, with the significance level of alpha = 0.05. Calculations of IC50 were made by the non-linear regression analysis in the Prism 5 software. If the IC50 was ambiguous it was reported as not applicable (N/A). If the suggested IC50 exceeded the highest tested concentration it was reported only if the R2 exceeded 0.75 or SI% for the highest concentration was under 75%, otherwise only defined as > highest tested concentration. An approximate (~) value was used as a true value when used to calculate cytotoxicity ratios. An unpaired two-tailed t-test was used to determine the significance levels of the ratios (p < 0.05, p < 0.01 and p < 0.001).
To verify hypoxia and anoxia in the cells, microarray analysis was performed as previously described  at the Uppsala Array Platform (Department of Medical Science, Science for Life Laboratory, Uppsala University, Sweden). MCF-7 breast cancer cells was incubated either in normoxic, hypoxic or anoxic surroundings, after 90 hours the cells were washed with PBS and total RNA was prepared using RNeasy® Mini Kit (Qiagen AB, Sollentuna, Sweden) according to the manufacturers instructions. RNA concentration was measured with ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and RNA quality was evaluated using the Agilent 2100 Bioanalyzer system (Agilent Technologies Inc, Palo Alto, CA). 250 ng of total RNA from each sample were used to generate amplified and biotinylated sense-strand cDNA from the entire expressed genome according to the Ambion WT Expression Kit (P/N 4425209 Rev C 09/2009) and Affymetrix GeneChip® WT Terminal Labeling and Hybridization User Manual (P/N 702808 Rev. 6, Affymetrix Inc., Santa Clara, CA). GeneChip® ST Arrays (GeneChip® Human Gene 2.0 ST Array) were hybridized for 16 hours in a 45°C incubator, rotated at 60 rpm. According to the GeneChip® Expression Wash, Stain and Scan Manual (PN 702731 Rev 3, Affymetrix Inc., Santa Clara, CA) the arrays were then washed and stained using the Fluidics Station 450 and finally scanned using the GeneChip® Scanner 3000 7G. The raw data was normalized in the free software Expression Console provided by Affymetrix (affymetrix.com) using the robust multi-array average (RMA) method. Further interpretation of the gene expression data was done by gene set enrichment analysis (GSEA)  and the gene ontology (GO) bioinformatic tool: database for annotation, visualization and integrated discovery (DAVID) .
The normoxic IC50-values for all drugs in the panel in the cell lines (A2780, ACHN, H69, MCF-7 and U-937) are shown in Table 2 and the IC50-ratios of hypoxic or anoxic vs normoxic cells are displayed in Table 3. If the ratio for a drug was close to 1 (arbitrarily set to 0.8-1.2), it was considered as equally effective in anoxic/hypoxic and normoxic cells. If the ratio exceeded 1.2 the effect of the drug was less effective in anoxia/hypoxia, and if the ratio was less than 0.8 the drug was more effective in anoxia/hypoxia.
Trends in the different cell lines
The ovarian carcinoma cell line A2780 was less sensitive to most drugs (ratio >1.2 in nine of 17 drugs evaluable for IC50) in anoxia (0.1% O2), but surprisingly was more or equally sensitive (ratio <1.2 in 14 of 16 drugs) to the administered drugs in hypoxia (1.0% O2) compared to normoxia. The renal adenocarcinoma ACHN was less sensitive to the effects of most drugs in both anoxic (ratio >1.2 in 10 of 12 drugs) and hypoxic cells (ratio >1.2 in 11 of 15 drugs) compared to normoxic cells. Compared to normoxic cells, oxygen deprived H69 (small lung cancer) and MCF-7 (breast cancer) cells were generally more sensitive to most drugs (for hypoxia the ratio was <0.8 in 11 of 13, and in 6 of 7 drugs respectively). U-937 (lymphoma) cells were slightly more, or equally, sensitive to most drugs in a hypoxic environment.
Trends between the different drugs
In general cisplatin, mitomycin c and tirapazamine (Figure 1) were more effective in anoxic or hypoxic environment (e.g. tirapazamine was significantly more active in all evaluated cell lines; cisplatin in H69, MCF-7 and U-937; and mitomycin C in A2780, H69, MCF-7 and U-937). Acriflavine, bortezomib, doxorubicin and etoposide also showed a slightly higher effect in anoxia and hypoxia compared to normoxia. Sorafenib and irinotecan (Figure 2) was apparently less effective in most anoxic and hypoxic cells (e.g. sorafenib was significantly less active in ACHN, MCF-7 and U-937), while docetaxel and melphalan had a slight decrease in effect in most anoxic and hypoxic cells. The other tested drug did not present with a clear tendency for being more or less sensitive in hypoxia or anoxia, the different cell types behaved differently (Table 3).
Sensitivity of untreated cells
The control/blank signal relationship between oxygen deprived and oxygenated cells were calculated to evaluate the proliferating abilities of the cells, since a diminished proliferative capacity is likely to render lower sensitivity to most cytotoxic drugs. The mean ratio of the control/blank signal of anoxic or hypoxic cells and normoxic cells are presented in Table 4. A value below 1 (a lower signal) indicates a lower cell number in control wells after 18 + 72 hrs incubation in oxygen deprived cells vs. normoxic cells, as would be expected theoretically. However, the comparably high cell density (100,000/mL, selected to give the best signal-noise ratio) and the 90 hrs total incubation will probably also lead to some extent of growth inhibition due to confluence and cell-cell inhibition in the normoxic cells during the experiment. In such cases it is possible that growth inhibition (i.e. cytostatic effects, in contrast to cell killing cytotoxic effects) in the end of the experiments may be underestimated. Low ratios were observed in ACHN, U-937 and anoxic A2780 cells, which appear to correlate with the lower sensitivity to most drugs in hypoxic/anoxic ACHN and anoxic A2780 cells. However, it appears that U-937 is the most sensitive cell line to oxygen deprivation in the panel, and this is not reflected by the changes in chemosensitivity. Surprisingly, a high ratio was observed in H69, and indeed this cell line was also generally more sensitive to most of the drugs tested. No significant discrepancy was observed in MCF-7, who still was slightly more sensitive to the drugs in hypoxia.
Gene set enrichment analysis shows a distinct pattern of hypoxia-associated gene sets among the genes up-regulated when incubated in hypoxia . Gene expression data confirmed that cells grown in oxygen-deprived surroundings to a higher degree expressed genes affiliated with hypoxia such as HIF1α (Figure 3). A clear pattern was also seen in the over-represented GO terms with the top result being the “response to hypoxia” (adjusted p-value 5.19E-13) group of 16 genes, also for the up-regulated genes. Raw and normalized expression data have been deposited at Gene Expression Omnibus with accession number GSE47009.
The concentration of oxygen in human tumors widely varies, and it is not uncommon to find areas with oxygen pressure lower than 2.5 mmHg, and the extent of hypoxia seems to be tumor stage and size independent . Radiotherapy and conventional chemotherapies are often less effective in oxygen depressed cells . Therefore it is of great importance to make use of the oxygen deprivation and find drugs that are more effective in hypoxic tumor cells.
In our study the untreated hypoxic and anoxic ACHN and U-937 cells, as well as anoxic A2780 cells were less proliferative than corresponding normoxic cells (i.e. most sensitive to oxygen deprivation). Indeed results also showed that ACHN and anoxic A2780 were more resistant to most drugs under reduced oxygen pressure, which is expected in view of the fact that slow proliferating tumor cells are less sensitive to chemotherapy. Interestingly the reversed effect could be observed in H69, where oxygen deprived cells (most surprisingly) appeared more viable and was a lot more sensitive to drugs. MCF-7 cells were also more sensitive to drugs in an oxygen-deprived environment but, in difference to H69, the MCF-7 cells displayed no proliferative difference in normoxic and hypoxic or anoxic surroundings. Hypoxia mostly occurs in tumors and therefore different cell lines with a solid tumor origin were the most interesting objects in this study. The leukemic lymphoma cell line U-937 is not a solid tumor per se, but was included in the study for comparison. Untreated U-937 cells were less viable in an oxygen-deprived environment, but did not display any real difference in sensitivity to chemotherapy in hypoxia or anoxia.
Three drugs were more effective in a hypoxic and anoxic environment; cisplatin, mitomycin c and tirapazamine. Earlier studies have revealed contradictive results, showing hypoxic cells to be more resistant to cisplatin in some cell lines  but also showing cisplatin to be a HIF-1 inhibitor . Mitomycin c was also clearly more effective in most of the oxygen deprived cell lines. Hypoxia induces the enzymatic system capable of activating mitomycin c  and is therefore considered more toxic to hypoxic cells [46, 54]. However, mitomycin c has also been shown to be less effective in hypoxic testicular germ cell tumor cell lines  and was in our study less effective in ACHN under hypoxic and anoxic conditions. Tirapazamine was significantly more effective in all oxygen deprived cell lines, and our results for tirapazamine highly correspond to previous studies of this bioreductive prodrug . Tirapazamine is activated under hypoxic conditions by a reductase enzyme, in which creating a highly reactive molecule that in turn causes single- and double strand breaks in the DNA of tumor .
The drugs with increased resistance in hypoxic and anoxic cells were docetaxel, irinotecan, melphalan and sorafenib. Docetaxel has been shown to both influence  and not influence  the HIF-1α protein accumulation. Although this study proposed that docetaxel was associated with increased drug resistance in most cells in anoxia and hypoxia, other studies has implied that some cell lines was not . In accordance to this study, irinotecan has earlier been shown to be less effective under hypoxic conditions . Irinotecan decreases the expression of HIF-1α and VEGF under both normoxic and hypoxic conditions , which could be why there is no difference in effect in some cell lines; here U-937. Melphalan is an alkylating agent with an enhanced effect in hypoxia  and in HIF-1α inhibited cells . Although the correlation between hypoxia and melphalan resistance was not distinct, both A2780 and ACHN were clearly less sensitive and U-937 more sensitive, in oxygen deprived cells. Sorafenib inhibits vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) signaling , thus one might hypothesize that sorafenib would be more potent under hypoxic conditions. With respect to the cell lines used in this report, we have found no information on SCLC cell line NCI-H69 expression or dependence on VEGF signaling. The renal cell adenocarcinoma ACHN has a low normal baseline secretion of VEGF to cell growth medium , a secretion that may be inhibited by sorafenib, and to which ACHN is sensitive . The breast cancer cell line MCF-7 has been described with a survival system by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR-1 , and cell line is sensitive to treatment with sorafenib, which also appear to down-regulate hypoxia induced HIF-1α expression . The ovarian carcinoma cell line A2780 expresses VEGFR-1 , but its sensitivity to sorafenib has not been described previously. In this study sorafenib was less effective in hypoxic and anoxic ACHN, MCF-7 and U-937 cells, which may be related to the mono-culture assay with no communicating stroma cells.
In the study presented herein we have emphasized to isolate hypoxia as the variable in the experiments, all other factors (nutrients in medium, cell density, incubation time etc.) were standardized, and all arms of each replicate (normoxic vs anoxic/hypoxic) were analyzed simultaneously. There are several environmental factors in solid tumors that may be studied, e.g. the low nutrient supply (analogous with oxygen supply), interaction with stroma cells, acidity (in part secondary to hypoxia, and metabolism), as well as proliferation of the tumor cells. These factors may be studied individually (as in this report), or by assays including several aspects, for example by the use of spheroid cultures or prolonged incubation times beyond confluency. Furthermore, since different drugs act on cancer cells in different ways resulting in cytostatic (growth inhibitory) or cytotoxic (cell killing) effects, different readouts would probably yield different results. The FMCA-based IC50-value used in this report is based on survival indices (compared to untreated control) at the end of the experiment, and is thus the result of both antiproliferative and toxic effects.
Our results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia. Part of the results obtained with this method, as probably with any model of oxygen deficiency, can be directly explained by decreased proliferation when cells are deprived of oxygen. However, this is clearly not the only variable, as some cells appeared to increase their proliferation and sensitivity under low oxygen pressure. Furthermore, hypoxia is not the only limiting factor of proliferation in a small tumor, but other limiting factors, such as the physical space, distribution of nutrients and drugs, metabolism and removal of waste products (with a succeeding change in pH), may also be utilized as therapeutic targets. These and other factors could also be evaluated in a similar screen study.
Durand RE, Raleigh JA: Identification of nonproliferating but viable hypoxic tumor cells in vivo. Cancer Res. 1998, 58 (16): 3547-3550.
Evans SM, Joiner B, Jenkins WT, Laughlin KM, Lord EM, Koch CJ: Identification of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide]. Br J Cancer. 1995, 72 (4): 875-882. 10.1038/bjc.1995.427.
Partridge SE, Aquino-Parsons C, Luo C, Green A, Olive PL: A pilot study comparing intratumoral oxygenation using the comet assay following 2.5% and 5% carbogen and 100% oxygen. Int J Radiat Oncol Biol Phys. 2001, 49 (2): 575-580. 10.1016/S0360-3016(00)01504-2.
Tannock IF: The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Br J Cancer. 1968, 22 (2): 258-273. 10.1038/bjc.1968.34.
Evans SM, Hahn SM, Magarelli DP, Koch CJ: Hypoxic heterogeneity in human tumors: EF5 binding, vasculature, necrosis, and proliferation. Am J Clin Oncol. 2001, 24 (5): 467-472. 10.1097/00000421-200110000-00011.
Hanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996, 86 (3): 353-364. 10.1016/S0092-8674(00)80108-7.
Zetter BR: Angiogenesis and tumor metastasis. Annu Rev Med. 1998, 49: 407-424. 10.1146/annurev.med.49.1.407.
Brahimi-Horn MC, Chiche J, Pouyssegur J: Hypoxia and cancer. J Mol Med. 2007, 85 (12): 1301-1307. 10.1007/s00109-007-0281-3.
Hicklin DJ, Ellis LM: Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol. 2005, 23 (5): 1011-1027.
Zhou J, Schmid T, Schnitzer S, Brune B: Tumor hypoxia and cancer progression. Cancer Lett. 2006, 237 (1): 10-21. 10.1016/j.canlet.2005.05.028.
Vander Heiden MG, Cantley LC, Thompson CB: Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009, 324 (5930): 1029-1033. 10.1126/science.1160809.
Cardone RA, Casavola V, Reshkin SJ: The role of disturbed pH dynamics and the Na+/H + exchanger in metastasis. Nat Rev Cancer. 2005, 5 (10): 786-795. 10.1038/nrc1713.
Bogenrieder T, Herlyn M: Axis of evil: molecular mechanisms of cancer metastasis. Oncogene. 2003, 22 (42): 6524-6536. 10.1038/sj.onc.1206757.
Grau C, Khalil AA, Nordsmark M, Horsman MR, Overgaard J: The relationship between carbon monoxide breathing, tumour oxygenation and local tumour control in the C3H mammary carcinoma in vivo. Br J Cancer. 1994, 69 (1): 50-57. 10.1038/bjc.1994.8.
Brizel DM, Dodge RK, Clough RW, Dewhirst MW: Oxygenation of head and neck cancer: changes during radiotherapy and impact on treatment outcome. Radiother Oncol. 1999, 53 (2): 113-117. 10.1016/S0167-8140(99)00102-4.
Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P: Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res. 1996, 56 (19): 4509-4515.
Bodner G, Schocke MF, Rachbauer F, Seppi K, Peer S, Fierlinger A, Sununu T, Jaschke WR: Differentiation of malignant and benign musculoskeletal tumors: combined color and power Doppler US and spectral wave analysis. Radiology. 2002, 223 (2): 410-416. 10.1148/radiol.2232010406.
Brown JM, Giaccia AJ: The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res. 1998, 58 (7): 1408-1416.
Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, Giaccia AJ: Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature. 1996, 379 (6560): 88-91. 10.1038/379088a0.
Brown JM, Wilson WR: Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer. 2004, 4 (6): 437-447. 10.1038/nrc1367.
Roninson IB: The role of the MDR1 (P-glycoprotein) gene in multidrug resistance in vitro and in vivo. Biochem Pharmacol. 1992, 43 (1): 95-102. 10.1016/0006-2952(92)90666-7.
Wartenberg M, Ling FC, Muschen M, Klein F, Acker H, Gassmann M, Petrat K, Putz V, Hescheler J, Sauer H: Regulation of the multidrug resistance transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species. FASEB J. 2003, 17 (3): 503-505.
Matzow T, Cowen RL, Williams KJ, Telfer BA, Flint PJ, Southgate TD, Saunders MP: Hypoxia-targeted over-expression of carboxylesterase as a means of increasing tumour sensitivity to irinotecan (CPT-11). J Gene Med. 2007, 9 (4): 244-252. 10.1002/jgm.1016.
Erler JT, Cawthorne CJ, Williams KJ, Koritzinsky M, Wouters BG, Wilson C, Miller C, Demonacos C, Stratford IJ, Dive C: Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance. Mol Cell Biol. 2004, 24 (7): 2875-2889. 10.1128/MCB.24.7.2875-2889.2004.
Lash GE, Fitzpatrick TE, Graham CH: Effect of hypoxia on cellular adhesion to vitronectin and fibronectin. Biochem Biophys Res Commun. 2001, 287 (3): 622-629. 10.1006/bbrc.2001.5618.
Kim JY, Kim YJ, Lee S, Park JH: The critical role of ERK in death resistance and invasiveness of hypoxia-selected glioblastoma cells. BMC Cancer. 2009, 9: 27-10.1186/1471-2407-9-27.
Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E: Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature. 1998, 394 (6692): 485-490. 10.1038/28867.
Griguer CE, Oliva CR, Kelley EE, Giles GI, Lancaster JR, Gillespie GY: Xanthine oxidase-dependent regulation of hypoxia-inducible factor in cancer cells. Cancer Res. 2006, 66 (4): 2257-2263. 10.1158/0008-5472.CAN-05-3364.
Baumann RP, Penketh PG, Seow HA, Shyam K, Sartorelli AC: Generation of oxygen deficiency in cell culture using a two-enzyme system to evaluate agents targeting hypoxic tumor cells. Radiat Res. 2008, 170 (5): 651-660. 10.1667/RR1431.1.
Minchinton AI, Tannock IF: Drug penetration in solid tumours. Nat Rev Cancer. 2006, 6 (8): 583-592. 10.1038/nrc1893.
Karlsson H, Fryknas M, Larsson R, Nygren P: Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system. Exp Cell Res. 2012, 318 (13): 1577-1585. 10.1016/j.yexcr.2012.03.026.
Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA: Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol. 2010, 148 (1): 3-15. 10.1016/j.jbiotec.2010.01.012.
Danielsen T, Skoyum R, Rofstad EK: Hypoxia-induced changes in radiation sensitivity in human melanoma cells: importance of oxygen-regulated proteins, adenylate energy charge and cell cycle distribution. Radiother Oncol. 1997, 44 (2): 177-182. 10.1016/S0167-8140(97)01929-4.
Yang SJ, Pyen J, Lee I, Lee H, Kim Y, Kim T: Cobalt chloride-induced apoptosis and extracellular signal-regulated protein kinase 1/2 activation in rat C6 glioma cells. J Biochem Mol Biol. 2004, 37 (4): 480-486. 10.5483/BMBRep.2004.37.4.480.
Koch S, Mayer F, Honecker F, Schittenhelm M, Bokemeyer C: Efficacy of cytotoxic agents used in the treatment of testicular germ cell tumours under normoxic and hypoxic conditions in vitro. Br J Cancer. 2003, 89 (11): 2133-2139. 10.1038/sj.bjc.6601375.
Hay MP, Gamage SA, Kovacs MS, Pruijn FB, Anderson RF, Patterson AV, Wilson WR, Brown JM, Denny WA: Structure-activity relationships of 1,2,4-benzotriazine 1,4-dioxides as hypoxia-selective analogues of tirapazamine. J Med Chem. 2003, 46 (1): 169-182. 10.1021/jm020367+.
Sakata K, Kwok TT, Murphy BJ, Laderoute KR, Gordon GR, Sutherland RM: Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance. Br J Cancer. 1991, 64 (5): 809-814. 10.1038/bjc.1991.405.
Rohwer N, Dame C, Haugstetter A, Wiedenmann B, Detjen K, Schmitt CA, Cramer T: Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB. PLoS One. 2010, 5 (8): e12038-10.1371/journal.pone.0012038.
Lee K, Zhang H, Qian DZ, Rey S, Liu JO, Semenza GL: Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization. Proc Natl Acad Sci USA. 2009, 106 (42): 17910-17915. 10.1073/pnas.0909353106.
Roccaro AM, Hideshima T, Raje N, Kumar S, Ishitsuka K, Yasui H, Shiraishi N, Ribatti D, Nico B, Vacca A, Dammacco F, Richardson PG, Anderson KC: Bortezomib mediates antiangiogenesis in multiple myeloma via direct and indirect effects on endothelial cells. Cancer Res. 2006, 66 (1): 184-191. 10.1158/0008-5472.CAN-05-1195.
Shin DH, Chun YS, Lee DS, Huang LE, Park JW: Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated repression of hypoxia-inducible factor-1. Blood. 2008, 111 (6): 3131-3136. 10.1182/blood-2007-11-120576.
Duyndam MC, van Berkel MP, Dorsman JC, Rockx DA, Pinedo HM, Boven E: Cisplatin and doxorubicin repress Vascular Endothelial Growth Factor expression and differentially down-regulate Hypoxia-inducible Factor I activity in human ovarian cancer cells. Biochem Pharmacol. 2007, 74 (2): 191-201. 10.1016/j.bcp.2007.04.003.
Zhang H, Qian DZ, Tan YS, Lee K, Gao P, Ren YR, Rey S, Hammers H, Chang D, Pili R, Dang CV, Liu JO, Semenza GL: Digoxin and other cardiac glycosides inhibit HIF-1alpha synthesis and block tumor growth. Proc Natl Acad Sci USA. 2008, 105 (50): 19579-19586. 10.1073/pnas.0809763105.
Escuin D, Kline ER, Giannakakou P: Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function. Cancer Res. 2005, 65 (19): 9021-9028. 10.1158/0008-5472.CAN-04-4095.
Forde JC, Perry AS, Brennan K, Martin LM, Lawler MP, Lynch TH, Hollywood D, Marignol L: Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells. Urol Oncol. 2010, 30: 912-919.
Kennedy KA, Siegfried JM, Sartorelli AC, Tritton TR: Effects of anthracyclines on oxygenated and hypoxic tumor cells. Cancer Res. 1983, 43 (1): 54-59.
Sermeus A, Cosse JP, Crespin M, Mainfroid V, de Longueville F, Ninane N, Raes M, Remacle J, Michiels C: Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity. Mol Cancer. 2008, 7: 27-
Sullivan R, Graham CH: Hypoxia prevents etoposide-induced DNA damage in cancer cells through a mechanism involving hypoxia-inducible factor 1. Mol Cancer Ther. 2009, 8 (6): 1702-1713. 10.1158/1535-7163.MCT-08-1090.
Onozuka H, Tsuchihara K, Esumi H: Hypoglycemic/hypoxic condition in vitro mimicking the tumor microenvironment markedly reduced the efficacy of anticancer drugs. Cancer Sci. 2011, 102 (5): 975-982. 10.1111/j.1349-7006.2011.01880.x.
Kasuya K, Tsuchida A, Nagakawa Y, Suzuki M, Abe Y, Itoi T, Serizawa H, Nagao T, Shimazu M, Aoki T: Hypoxia-inducible factor-1alpha expression and gemcitabine chemotherapy for pancreatic cancer. Oncol Rep. 2011, 26 (6): 1399-1406.
Kamiyama H, Takano S, Tsuboi K, Matsumura A: Anti-angiogenic effects of SN38 (active metabolite of irinotecan): inhibition of hypoxia-inducible factor 1 alpha (HIF-1alpha)/vascular endothelial growth factor (VEGF) expression of glioma and growth of endothelial cells. J Cancer Res Clin Oncol. 2005, 131 (4): 205-213. 10.1007/s00432-004-0642-z.
de Wilt JH, Manusama ER, van Tiel ST, van Ijken MG, ten Hagen TL, Eggermont AM: Prerequisites for effective isolated limb perfusion using tumour necrosis factor alpha and melphalan in rats. Br J Cancer. 1999, 80 (1–2): 161-166.
Hu Y, Kirito K, Yoshida K, Mitsumori T, Nakajima K, Nozaki Y, Hamanaka S, Nagashima T, Kunitama M, Sakoe K, Komatsu N: Inhibition of hypoxia-inducible factor-1 function enhances the sensitivity of multiple myeloma cells to melphalan. Mol Cancer Ther. 2009, 8 (8): 2329-2338. 10.1158/1535-7163.MCT-09-0150.
Teicher BA, Lazo JS, Sartorelli AC: Classification of antineoplastic agents by their selective toxicities toward oxygenated and hypoxic tumor cells. Cancer Res. 1981, 41 (1): 73-81.
Hudson CC, Liu M, Chiang GG, Otterness DM, Loomis DC, Kaper F, Giaccia AJ, Abraham RT: Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin. Mol Cell Biol. 2002, 22 (20): 7004-7014. 10.1128/MCB.22.20.7004-7014.2002.
Mayerhofer M, Valent P, Sperr WR, Griffin JD, Sillaber C: BCR/ABL induces expression of vascular endothelial growth factor and its transcriptional activator, hypoxia inducible factor-1alpha, through a pathway involving phosphoinositide 3-kinase and the mammalian target of rapamycin. Blood. 2002, 100 (10): 3767-3775. 10.1182/blood-2002-01-0109.
Pencreach E, Guerin E, Nicolet C, Lelong-Rebel I, Voegeli AC, Oudet P, Larsen AK, Gaub MP, Guenot D: Marked activity of irinotecan and rapamycin combination toward colon cancer cells in vivo and in vitro is mediated through cooperative modulation of the mammalian target of rapamycin/hypoxia-inducible factor-1alpha axis. Clin Cancer Res. 2009, 15 (4): 1297-1307. 10.1158/1078-0432.CCR-08-0889.
Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, Wilhelm S, Lynch M, Carter C: Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 2006, 66 (24): 11851-11858. 10.1158/0008-5472.CAN-06-1377.
D’Amato RJ, Loughnan MS, Flynn E, Folkman J: Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci USA. 1994, 91 (9): 4082-4085. 10.1073/pnas.91.9.4082.
Bauer KS, Dixon SC, Figg WD: Inhibition of angiogenesis by thalidomide requires metabolic activation, which is species-dependent. Biochem Pharmacol. 1998, 55 (11): 1827-1834. 10.1016/S0006-2952(98)00046-X.
Brown JM: The hypoxic cell: a target for selective cancer therapy–eighteenth Bruce F Cain Memorial Award lecture. Cancer Res. 1999, 59 (23): 5863-5870.
Marcu L, Olver I: Tirapazamine: from bench to clinical trials. Curr Clin Pharmacol. 2006, 1 (1): 71-79. 10.2174/157488406775268192.
Reddy SB, Williamson SK: Tirapazamine: a novel agent targeting hypoxic tumor cells. Expert Opin Investig Drugs. 2009, 18 (1): 77-87. 10.1517/13543780802567250.
Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, Melillo G: Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res. 2002, 62 (15): 4316-4324.
Kummar S, Raffeld M, Juwara L, Horneffer Y, Strassberger A, Allen D, Steinberg SM, Rapisarda A, Spencer SD, Figg WD, Chen X, Turkbey IB, Choyke P, Murgo AJ, Doroshow JH, Melillo G: Multihistology, target-driven pilot trial of oral topotecan as an inhibitor of hypoxia-inducible factor-1alpha in advanced solid tumors. Clin Cancer Res. 2011, 17 (15): 5123-5131. 10.1158/1078-0432.CCR-11-0682.
Liu L, Ning X, Sun L, Zhang H, Shi Y, Guo C, Han S, Liu J, Sun S, Han Z, Wu K, Fan D: Hypoxia-inducible factor-1 alpha contributes to hypoxia-induced chemoresistance in gastric cancer. Cancer Sci. 2008, 99 (1): 121-128.
Lindhagen E, Nygren P, Larsson R: The fluorometric microculture cytotoxicity assay. Nat Protoc. 2008, 3 (8): 1364-1369. 10.1038/nprot.2008.114.
Larsson R, Kristensen J, Sandberg C, Nygren P: Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA). Int J Cancer. 1992, 50 (2): 177-185. 10.1002/ijc.2910500204.
Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, Lerner J, Brunet JP, Subramanian A, Ross KN, Reich M, Hieronymus H, Wei G, Armstrong SA, Haggarty SJ, Clemons PA, Wei R, Carr SA, Lander ES, Golub TR: The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science. 2006, 313 (5795): 1929-1935. 10.1126/science.1132939.
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005, 102 (43): 15545-15550. 10.1073/pnas.0506580102.
da Huang W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009, 4 (1): 44-57.
Elvidge GP, Glenny L, Appelhoff RJ, Ratcliffe PJ, Ragoussis J, Gleadle JM: Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the role of HIF-1alpha, HIF-2alpha, and other pathways. J Biol Chem. 2006, 281 (22): 15215-15226. 10.1074/jbc.M511408200.
Vaupel P, Thews O, Hoeckel M: Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol. 2001, 18 (4): 243-259. 10.1385/MO:18:4:243.
Rohwer N, Cramer T: Hypoxia-mediated drug resistance: novel insights on the functional interaction of HIFs and cell death pathways. Drug Resist Updat. 2011, 14 (3): 191-201. 10.1016/j.drup.2011.03.001.
Schwartz HS: Pharmacology of mitomycin C. III. In vitro metabolism by rat liver. J Pharmacol Exp Ther. 1962, 136: 250-258.
Iwai A, Fujii Y, Kawakami S, Takazawa R, Kageyama Y, Yoshida MA, Kihara K: Down-regulation of vascular endothelial growth factor in renal cell carcinoma cells by glucocorticoids. Mol Cell Endocrinol. 2004, 226 (1–2): 11-17.
Tochizawa S, Masumori N, Yanai Y, Ohmoto Y, Yabuuchi Y, Tsukamoto T: Antitumor effects of a combination of interferon-alpha and sorafenib on human renal carcinoma cell lines. Biomed Res. 2008, 29 (6): 271-278. 10.2220/biomedres.29.271.
Lee TH, Seng S, Sekine M, Hinton C, Fu Y, Avraham HK, Avraham S: Vascular endothelial growth factor mediates intracrine survival in human breast carcinoma cells through internally expressed VEGFR1/FLT1. PLoS Med. 2007, 4 (6): e186-10.1371/journal.pmed.0040186.
Lee JH, Shim JW, Choi YJ, Heo K, Yang K: The combination of sorafenib and radiation preferentially inhibits breast cancer stem cells by suppressing HIF-1alpha expression. Oncol Rep. 2013, 29 (3): 917-924.
Spannuth WA, Nick AM, Jennings NB, Armaiz-Pena GN, Mangala LS, Danes CG, Lin YG, Merritt WM, Thaker PH, Kamat AA, Han LY, Tonra JR, Coleman RL, Ellis LM, Sood AK: Functional significance of VEGFR-2 on ovarian cancer cells. Int J Cancer. 2009, 124 (5): 1045-1053. 10.1002/ijc.24028.
The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/331/prepub
This work was supported by Research Fund at the Department of Oncology, Uppsala University Hospital (Stiftelsen Onkologiska Klinikens i Uppsala Forskningsfond), and the LIONS Cancer Research fund.
Lena Lenhammar, Nasrin Najafi and Emelie Larsson are gratefully acknowledged for skillful technical assistance.
The authors declare that they have no competing interests.
SS individually performed all experimental work, collected and processed raw data, and drafted the manuscript. MF is appointed co-supervisor, participated in the experimental design and in particular interpretation of microarray analysis. RL is appointed co-supervisor and head of department, RL participated in the design of the study and interpretation of data. JG is appointed main supervisor, conceived of the study, participated in its design and co-ordination with co-workers/authors. JG was also involved in analyzing and interpretation data yielded, and had an active role in drafting the manuscript together with SS. All authors read and approved the final manuscript.
About this article
Cite this article
Strese, S., Fryknäs, M., Larsson, R. et al. Effects of hypoxia on human cancer cell line chemosensitivity. BMC Cancer 13, 331 (2013). https://doi.org/10.1186/1471-2407-13-331
- Cancer cell lines
- Hypoxic incubator
- Drug resistance