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Effect of hypoxia on chemosensitivity to 5-fluorouracil in SH-SY5Y neuroblastoma cells

Effect of hypoxia on chemosensitivity to 5-fluorouracil in SH-SY5Y neuroblastoma cells BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw005 Research article Eec ff t of hypoxia on chemosensitivity to 5-fluorouracil in SH-SY5Y neuroblastoma cells Hannah Rose Warren* and Momna Hejmadi Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA1 7AY, England *Corresponding author: Email: hannah.r.warren@bath.edu Supervisor: Momna Hejmadi, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA1 7AY, UK. Resistance to chemotherapy is a major obstacle in clinical oncology. Hypoxia is a hallmark of solid tumours as a result of poorly structured tumour neovasculature and is known to be a key contributor to cancer malignancy and reduced drug effectiveness. Hypoxia-inducible factor-1 (HIF-1) mediates the hypoxic response, bringing about adaptive responses to the change in micro- environment. This study investigated the effect of 24 h hypoxia on the sensitivity of SH-SY5Y neuroblastoma cells to 5-fluoro - uracil (5-FU). Cytotoxicity, apoptosis and membrane integrity cell-based assays were carried out to observe changes in cell viability alongside apoptotic and necrotic cell death to determine the molecular mechanisms underlying reduced drug sensi- tivity. Hypoxic cells showed no change in cell viability to 5-FU in comparison to normoxic controls, suggesting that hypoxia conferred reduced drug sensitivity. As a measure of apoptosis, caspase-3/7 levels were significantly higher in hypoxic cells treated with increasing 5-FU concentrations. However, this was associated with less cell necrosis, suggesting that despite increased hypoxia-induced apoptosis, a significant decrease in uncontrolled cell death accounted for the change in viability observed. This finding also implies that alternative cell survival mechanisms could also be activated. Key words: hypoxia, HIF-1, 5-fluorouracil, SH-SY5Y, apoptosis, necrosis Submitted on 7 September 2015; accepted on 6 June 2016 Introduction The hypoxic response is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor. HIF-1 is Hypoxia, a reduction in tissue oxygen levels, is a hallmark of composed of an oxygen-sensitive alpha subunit (HIF-1α) and solid tumour micro-environments and drastically effects a constitutively expressed beta subunit (HIF-1β). Under nor- tumour sensitivity to chemotherapy (Harris, 2002; Tredan moxic conditions, HIF-1α subunits are unstable due to et al., 2007). Regions of hypoxia are associated with distinct hydroxylation of specific proline residues regulated by HIF alterations in the tumour micro-environment, such as increas- prolyl-hydroxylase domain (PHD) proteins, promoting asso- ing acidosis as a response of metabolic switching to glycolysis, ciation with the Von Hippel-Lindau protein (pVHL) ubiquitin reduced nutrient availability and interstitial fluid pressure E3 ligase and subsequent destruction by the proteasome. (Tannock et al., 2002; Brahimi-Horn et al., 2007). Tumours Under hypoxia, HIF-1α is stabilized by the inhibition of PHDs often show a central core of necrotic cells where severe oxygen and instead translocates to the nucleus where it dimerizes with deprivation results in cell death (Brahimi-Horn et al., 2007). HIF-1β. Following recruitment of co-activators, the complex Despite increased angiogenesis, the demand for oxygen always formed binds to the hypoxia response element (HRE) and exceeds the supply, causing hypoxia to remain a constant fea- induces transcription of target genes. The contribution of ture. To survive, tumour cells adapt to these conditions by HIF-1 to drug resistance is complex and has been observed in changes in gene expression, which along with the limited deliv- a range of solid tumours (Sasabe et al., 2007; Liu et al., 2008; ery of drugs to these cells increases resistance to chemotherapy Sullivan et  al., 2008). Active HIF-1 targets ∼1–2% of the and promotes a malignant and more aggressive phenotype. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Research article Bioscience Horizons • Volume 9 2016 human genome, differentially regulating genes involved in a metabolism was shown to confer resistance to RIP-dependent number of biological processes such as angiogenesis, cellular necroptosis via suppression of mitochondrial ROS (Huang metabolism, pH regulation, cell survival and cell death path- et al., 2013). Interestingly, BNIP3 up-regulated under hypoxia ways (Brahimi-Horn et al., 2007). can also mediate a necrosis-like cell death by opening of mito- chondrial pores causing mitochondrial dysfunction independent Cell death and survival pathways under of cytochrome-c release (Vande-Velde et al., 2000). However, this response is thought to be induced only by severe acidosis hypoxia associated with chronic hypoxia (Pouysségur et al., 2006). Hypoxia can induce cell death by a number of distinct path- ways although its effects can be cell type dependent and differ Hypoxia-induced resistance to 5-flurouracil according to severity (Kilic et  al., 2007; Pakravan, 2013). The chemotherapeutic agent 5-fluorouracil (5-FU) is an anti- Severe hypoxia initiates apoptotic or necrotic cell death, metabolite that exerts its cytotoxic effects through the inhibi- whereas under acute milder hypoxia some cells can escape tion of thymidylate synthase and misincorporation into DNA these cell death pathways and survive (Zhou et  al., 2006). inhibiting DNA synthesis (Longley et al., 2003). Mechanisms However, moderate levels of hypoxia can manifest an array of of hypoxia-induced resistance to 5-FU have been reported to responses from an imbalance of cell survival or cell death fac- arise from increased drug efflux expression, G1 phase cycle tors, thus highlighting the inconclusive nature of transient arrest and induced senescence, and inhibition of apoptosis hypoxia within a tumour (Harris, 2002; Cosse and Michiels, (Chang et al., 2006; Sasabe et al., 2007; Yoshiba et al., 2009; 2008; Qiu et al., 2015). Rohwer et al., 2010). However, following DNA damage by Apoptosis is regulated by the members of the Bcl-2 (B-cell chemotherapeutic agents, other cellular responses can be pro- lymphoma 2) protein family which act as positive and negative moted such as autophagy, necrosis and mitotic catastrophe, regulators. Death factors include Bax and Bak which upon acti- suggesting that suppression of a single death pathway may be vation associate with the mitochondrial membrane and cause inadequate to promote survival (Sullivan et al., 2008). This membrane permeabilization releasing cytochrome-c into the study aims to understand how hypoxia influences the sensitiv - cytoplasm. Cytochrome-c binds to Apaf-1 (apoptotic protease ity of SH-SY5Y neuroblastoma cells to 5-fluorouracil, by activating factor 1), which activates caspase 9, in turn cleaving determining changes to the prevalence and type of cell death. caspases 3 and 6. Their activation and cleavage generates a con- trolled signalling cascade permitting cellular changes such as Materials and methods membrane blebbing and programmed disassembly of the cell. Anti-apoptotic proteins such as Bcl-2 and Bcl-xL can shift the All standard reagents were purchased from Fisher Scientific balance to cell survival by inhibiting the function of Bax and unless stated otherwise. Bak. HIF-1α can induce apoptosis by overexpression of pro- apoptotic proteins such as BNIP3 (Bcl-2/adenovirus E1B 19 kDa SH-SY5Y cell line interacting protein 3) and its homologue BNIP3L (NIX) which SH-SY5Y neuroblastoma cells were obtained from American inhibit Bcl-2 and Bcl-xL (Sowter et al., 2001). HIF-1 also pro- Tissue Culture Collection (ATCC) and authenticated using motes p53-dependent apoptosis by increasing the stability of the their testing service. Cells were maintained in Dulbecco’s p53 tumour suppressor gene (Schmid et al., 2004; Zhou et al., Modified Eagle Medium (DMEM-F12 Reduced Serum) 2015). The mutational status of p53 is an important factor as (GIBCO, Invitrogen, Paisley, UK), plus 10% (v/v) fetal bovine loss of functional p53 prevents hypoxia-induced apoptosis serum, 100 µ g penicillin/streptomycin and 0.1 mM l-gluta- (Graeber et al., 1996; An et al., 1998; Sermeus and Michiels, mine. Cells were incubated at 37°C 5% CO and passaged to 2011). Tumour cells can avoid hypoxia-induced apoptosis by 2 maintain optimum confluence for experiments. The cell line up-regulation of anti-apoptotic factors in the Bcl-2 family, along was routinely tested for mycoplasma after 25 passages. with HIF-1-independent pathways such as inhibitor of apopto- sis protein 2 (IAP2) (Dong et al., 2003). Furthermore, in colon Hypoxia exposure cancer cells, the down-regulation of pro-apoptotic factors Bid, Bax and Bad was shown to increase drug resistance, implying Cells were seeded in 96-well plates at a density of 10 cells per protection from apoptosis (Erler et al., 2004). well (in 100 µ l medium) and cultured 24 h prior to use. Concentrations of 5-FU were diluted in culture medium and In contrast, necrosis is characterized by loss of cytoplasmic added to wells. Cells were then incubated under normoxic or integrity, cytoplasmic swelling and nuclear pyknosis, although hypoxic (5% CO , 95% N ) conditions at 37°C for 24 h. 2 2 its biochemical pathways are less well understood. Some necro- sis is regulated termed ‘necroptosis’ by alternative pathways In vitro cytotoxicity by MTT assay involving receptor-interacting protein kinase 1 and 3 (RIP1/ RIP3) which shift cell death from apoptosis to necrosis (Zhang Following exposure to 5-FU, cell viability was determined et al., 2009). Caspase inhibition, ATP availability and mitochon- using an MTT assay. The yellow tetrazolium salt MTT drial generation of reactive oxygen species (ROS) enhance this (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bro- switch (Nikoletopoulou et al., 2013). Under hypoxia, glycolytic mide) is reduced by metabolically active cells by dehydroge- 2 Bioscience Horizons • Volume 9 2016 Research article nase enzymes found in the mitochondria and converted into bated at room temperature and luminescence was measured at an insoluble purple formazan. Isopropanol can be used to dis- time 0, 30 and 60 min after the start of the assay, using a solve the formazan product and its absorbance measured to Promega GloMax plate reader (Promega) at 499 nm (excita- estimate the percentage of viable cells. One milligram per mil- tion) and 521 nm (emission). lilitre of MTT was dissolved in culture medium. Medium was Membrane integrity assay removed from 96-well plates, replaced by 50 µl of MTT solu - tion and incubated for 30 min at 37°C. MTT solution was The CytoTox-ONE™ Homogenous Membrane Integrity removed and the formazan product dissolved in 100 µ l of Assay (Promega) was used to measure the release of lactate isopropanol per well. Absorbance at 560 nm was measured dehydrogenase (LDH) from cells with a damaged membrane using a Modulus Microplate Reader (Turner Biosciences, and was therefore used to indicate the amount of cell necrosis. Promega, Southampton, UK). Cells were plated in 96-well plates as above, with quadruple replicates per plate (normoxic and hypoxic). Following Apoptosis assay hypoxic treatment, 2 µ l of lysis buffer was added to designated The Apo-ONE Homogeneous Caspase-3/7 Assay (Promega) wells containing cells for each treatment plate to act as a max- was used to measure the activity of caspase-3 and −7, the key imum LDH control. Forty microlitres of medium was then effectors of apoptosis. Cells were plated in 96-well plates as transferred to a new 96-well plate (the remaining cells used for before with triplet repeats per plate (normoxic and hypoxic). the Caspase-3/7 assay). Following the manufacturer’s instruc- The protocol was carried out according to the manufacturer’s tions, and working in the dark, one vial of substrate was mixed instructions. Working in the dark, 10 µ l of 100X substrate with 11 ml of buffer. Forty microlitres of assay reagent was was added to 990 µl of buffer. Following hypoxic exposure, a added to each well of transferred medium and incubated at 1:1 ratio using 40 µl of assay reagent was added to each well room temperature for 10 min. Twenty microlitres of stop solu- containing 40 µ l of sample medium. The plates were incu- tion was then added to each well, and the florescence was Figure 1. Effect of hypoxic treatment on cell viability, caspase-3/7 activity and membrane integrity. SHSY-5Y cells were exposed to normoxic and hypoxic conditions for 24 h. (A) Cell viability was measured by MTT assay. Data are expressed as percentage of normoxic controls, and values are represented as means ± SEM from four independent experiments, each containing eight replicates per condition. *Statistical significance using t-test at p < 0.001 (compared with normoxic control). (B) Apoptosis levels were measured by caspase-3/7 activity. Data are expressed as percentages of normoxic controls, and values are presented as means ± SEM from two independent experiments with three replicates per condition at each time interval. *Statistical significance using two-way ANOVA at p < 0.05. (C) Membrane integrity was measured by LDH concentration in the medium. Data are expressed as percentage of maximum normoxic LDH release from lysed cells, and values are presented as means ± SEM from two independent experiments with eight replicates per condition. 3 Research article Bioscience Horizons • Volume 9 2016 Figure 2. Effect of hypoxia and 5-fluorouracil on cell viability, caspase-3/7 activity and membrane integrity. SHSY-5Y cells were exposed to normoxic and hypoxic conditions for 24 h with varying concentrations (0–400 µM) of 5-FU. All statistical tests carried out with two-way ANOVA. (A) Cell viability was measured by MTT assay. Data are expressed as percentages of normoxic no drug control, and values are presented as means ± SEM from three to four independent experiments with eight replicates per treatment. *Statistical significance at p < 0.001. (B) Normoxic apoptosis levels were measured by caspase-3/7 activity taken at time intervals (0, 30 and 60 min after the start of the normoxic assay). Data are expressed as percentage of normoxic no drug controls, and values are presented as means ± SEM from two independent experiments with three replicates per treatment at each time interval. *Statistical significance at p < 0.05 and **at p < 0.001. (C) Apoptosis levels were measured by caspase-3/7 activity taken at 60 min after the start of the assay. Data are expressed as percentage of normoxic control and values represented as means ± SEM from two independent experiments with three replicates per treatment. *Statistical significance at p < 0.05. (D) Membrane integrity was measured by LDH concentration in the medium. Data are expressed as percentage of maximum LDH release from lysed cells and values shown as means ± SEM from two independent experiments with eight replicates per treatment. ® ® measured on a Promega GloMax plate reader (Promega) at were carried out using GraphPad Prism version 6.05 560 nm (excitation) and 590 nm (emission). (GraphPad Software, La Jolla, CA, USA). Statistical analysis Results Data are presented as means ± SEM. Statistical significance was determined using t-tests and one-way/two-way ANOVA The effect of hypoxia and 5-FU on cell viability, apoptosis and with Dunnetts post-hoc as stated. Graphs and statistical tests necrosis was analysed using cell-based assays mentioned above. 4 Bioscience Horizons • Volume 9 2016 Research article Table 1. Hypoxia and 5-FU statistical analysis Initial experiments looked at the effect of hypoxia alone on cell viability, caspase-3/7 activity and membrane integrity. SH-SY5Y cells were incubated in normoxic and hypoxic con- Two-way Source of % of total p-value Significant? ANOVA variation variation ditions for 24 h. Following treatment, there was a significant difference in cell viability between normoxic and hypoxic cells Condition 1.284 0.0124 Yes using the MTT assay (Fig. 1A). Cells treated with hypoxia had 50% reduction in cell viability. Hypoxic data also show a sig- MTT 5-FU 6.086 <0.0001 Yes nificant difference in caspase-3/7 activity compared with nor - Interaction 3.401 0.0057 Yes moxic controls but only after 60 min of assay incubation (Fig.  1B). This suggests that hypoxic cells underwent more Condition 21.68 0.0012 Yes apoptosis than normoxic cells. However, there was no signifi - Caspase cant variation in LDH release between normoxic and hypoxic 5-FU 5.425 0.3926 No 3/7 cells, suggesting no changes in necrosis (Fig. 1C). Interaction 3.074 0.6316 No The effect of 5-FU alone on SH-SY5Y cells was determined Condition 5.586 0.008 Yes using a range of drug concentrations (0–400 µ M) under nor- moxic conditions for 24 h. The high concentration of 5-FU LDH 5-FU 4.259 0.1417 No was determined by previous studies using SH-SY5Y cells and 5-FU (Tieu et al., 1999) as well as Phase II studies of 5-FU in Interaction 1.163 0.679 No other cancer cell lines (Chua et al., 2000; Kim et al., 2003). Results from the MTT assay show that cell viability signifi - cantly decreases with the addition of 5-FU, although this Both normoxic and hypoxic treated cells show a general effect is reduced with increasing concentrations of 5-FU trend of increased LDH release with increasing 5-FU concen- (Fig. 2A). Analysis by two-way ANOVA of caspase-3/7 activ- tration (Fig. 2D), and further analysis by two-way ANOVA ity shows that there is a significant difference in levels of showed that the overall variation between the two conditions apoptosis with the addition of 5-FU compared with the no is significant (Table  1). This supports that hypoxia has drug control (Fig.  2B). However, the level of significance decreased levels of necrotic cell death in comparison. decreases at 400 µ M 5-FU, suggesting that an alternative cell death mechanism could be accounting for the decreased cell viability seen in the MTT assay (Fig. 2A) at this concentra- Discussion tion. Alternatively, the LDH assay shows a trend of increasing Eec ff t of hypoxia and 5-flurouracil on cell LDH release with increasing 5-FU, implying that there is increased necrosis with higher concentrations of 5-FU viability (Fig. 2D). This study shows that SH-SY5Y cells treated with 5-FU under To determine the effect of hypoxia on chemosensitivity, 24 h hypoxia showed reduced sensitivity to 5-FU. As shown SH-SY5Y cells were treated with both varying concentrations by MTT, the addition of 5-FU to oxygenated cells signifi - of 5-FU (0–400 µ M) and 24 h hypoxia. In comparison to cantly reduced cell viability, whereas in hypoxia there was no normoxic conditions, the addition of 5-FU had no significant change in cell viability with increasing 5-FU concentrations. effect on cell viability under hypoxia (Fig. 2A). Furthermore, Similar results of reduced 5-FU sensitivity under hypoxia no significant difference was seen in cell viability between have been previously identified in other cancer cell lines such normoxic and hypoxic cells treated with individual 5-FU as oral squamous cell carcinoma (Yoshiba et al., 2009), gas- concentrations. Statistical analysis using two-way ANOVA tric cancer (Liu et al., 2009) and liver cancer (Yu et al., 2015). revealed the interaction of hypoxia and 5-FU to be signifi - A single 24 h hypoxic treatment was decided upon due to no cant, overall supporting that hypoxia has a significant effect significant variation in HeLa cell viability between normoxic, on reducing the sensitivity of SH-SY5Y cells to 5-FU acute (4 h hypoxia) and intermittent (4 h hypoxia per day for (Table 1). 2 days) hypoxic treatments, and chronic hypoxia (72 h) was toxic to cells (Pool, 2012). However, this 24 h treatment Previous results showed that only after 60 min a signifi - might still not be long enough to see a reduction in cell viabil- cant difference in caspase-3/7 activity could be detected ity using the MTT assay, and therefore, a later time point of between normoxic and hypoxic cells (Fig. 1B). At this same 48 h could also be investigated (Yu et al., 2015). time point, the addition of increasing 5-FU shows a general trend that cells treated with hypoxia had increasing cas- Despite our co-exposure to hypoxia and 5-FU showing a pase-3/7 activity in comparison to normoxic conditions, difference in viability, it would be interesting to see how pre- although this was only statistically significant at 400 µ M exposure to hypoxia, as would be the case in vivo, alters this 5-FU (Fig. 2C). Two-way ANOVA showed that the change in observation. SH-SY5Y cells pre-incubated under hypoxia for condition, rather than 5-FU, has a significant role in trigger - 23 h before drug treatment showed increased viability and a ing apoptosis (Table 1). more resistant phenotype (Hussein et al., 2006). 5 Research article Bioscience Horizons • Volume 9 2016 Figure 3. Hypoxic cell death and cell survival pathways. An overview of different cell responses under varying hypoxia. Hypoxia can promote cell survival by activation of HIF-1-independent pathways such as the PI3K/AKT or Ras/ERK pathways which induce adaptation to the hypoxic micro- environment. ERK can bind to HIF-1α to activate its transcriptional activity. Adaptive responses include up-regulation of angiogenic factors (VEGF), glucose and lactate transporters (GLUT-1, MCT-4), anti-apoptotic factors (Bcl-2, Bcl-XL) and drug efflux pumps (MDR-1) (Brahimi-Horn et al., 2007). Under conditions with low nutrient and ATP availability, hypoxia can inhibit mTOR activity, reducing protein synthesis to conserve energy. Low mTOR activity favours autophagy as a cell survival response which can provide a stress-resistant state. HIF-1 regulation of BNIP3 is involved in activation of autophagy. However, severe hypoxia can induce autophagic cell death. When ATP is conserved, HIF-1 can up-regulate p53, BNIP3 and NIX to promote apoptosis via Bax/Bak activation and subsequent activation of the caspase cascade. Apoptotic cells can undergo secondary necrosis. Necroptosis is an alternative programmed cell death that is independent of caspase activity and is activated by RIP1/RIP3 signalling via death receptors in the absence of caspases (Zhang et al., 2009). When cells are depleted of energy resources under hypoxia, necrosis can be activated by increasing ROS and HIF-1 up-regulation of BNIP3 which causes mitochondrial pore opening. susceptibility to hypoxia-induced apoptosis (Rössler et  al., Eec ff t of hypoxia and 5-flurouracil 2001; Qing et al., 2010). Hypoxia also enhanced the effect of on  apoptosis and necrosis flavopiridol, a cyclin-dependent kinase inhibitor, increasing the percentage of cells undergoing apoptosis compared with Under normoxic conditions, the addition of 5-FU significantly the drug alone (Puppo et al., 2004). Hypoxia-induced apopto- increased the level of apoptosis. At the higher 5-FU, concen- sis in neuroblastoma has been attributed to stabilization of tration of 400 µ M caspase activity decreased which was p53 and caspase activation (Araya et al., 1998; Chen et al., accompanied by increasing LDH release and necrosis. This 2003). The mechanisms accounting for the reduced sensitivity indicates a switch from apoptosis to necrosis with increasing of SH-SY5Y to 5-FU under hypoxia, despite increased apop- 5-FU, suggesting the occurrence of dose-dependent necrosis or tosis, are unclear. It is important to consider that long-term secondary necrosis (Guchelaar et  al., 1998). Hypoxic cells culture increases associated chromosomal abnormalities and showed higher levels of apoptosis after 60 min which that N-myc overexpressing populations could result in pre- increased with the addition of 5-FU, and at 400 µM, this was dominant apoptosis under hypoxia, while cells with normal significantly higher than normoxic cells. This was associated N-myc survive (Rössler et al., 2001; Ushmorov et al., 2008; with reduced overall LDH release, implying that there is less Poljaková et al., 2014). necrosis and more apoptosis. Interestingly, in a previous report, acute (4 h) and intermit- Similar results were reported in other neuroblastoma cell tent (4 h per day for 2 days) hypoxia showed significantly lines where amplification of the N-myc oncogene increased 6 Bioscience Horizons • Volume 9 2016 Research article lower levels of apoptosis in HeLa cells compared with nor- Conclusion moxic conditions (Pool, 2012). Our 24 h hypoxia is more severe and sustained, highlighting the differential role of vary- This study has shown that hypoxia reduces sensitivity to 5-FU ing hypoxia on cell death mechanisms. Neuroblastoma cells in a neuroblastoma cell line. Despite increased apoptosis, cell are also inherently different in terms of gene expression and viability remained unchanged with increasing 5-FU concen- sensitivity to hypoxia due to different rates of cell growth and trations, suggesting that along with decreased necrosis other cell death (Jögi et al., 2004). cell survival mechanisms must be in place. Further work is needed to confirm the exact mechanisms involved in reduced Alternatively, hypoxia can trigger other survival mecha- drug sensitivity of SH-SY5Y cells under hypoxia. To provide a nisms, which alongside decreased necrotic cell death, must comprehensive view of chemoresistance under hypoxia, other play a role in maintaining the cell viability and reduced drug mechanisms of cell death and survival beside apoptosis and sensitivity observed. Hypoxia can promote cellular adapta- necrosis should be investigated. Autophagy is known to have tion and survival by suppression of protein synthesis and a role during hypoxia, and assays would provide an insight cell growth via mTOR (mammalian target of rapamycin) into its adaptive or detrimental role under hypoxia. (Arsham et al., 2003; Pouysségur et al., 2006). Low mTOR activity under hypoxia favours the activation of autophagy, a survival response to remove damaged or unwanted organ- Author’s biography elles to preserve cell fitness and oxygen homeostasis ( Zhang H.R.W. graduated from the University of Bath in 2015 with a et  al., 2008). Here autophagy is considered protective first class honours degree in BSc Molecular and Cellular against apoptosis and can provide a stress-resistant state Biology. This research was carried out as part of her final-year supporting chemotherapeutic resistance (Chen et al., 2010). dissertation project and was chosen due to her interest in the Functional inhibition of autophagy in colon cancer cells was mechanisms of cancer drug resistance. Other fields of interest shown to restore chemosensitivity to 5-fluorouracil and include clinical pathology, infection and immunity and micro- increase tumour cell death (Li et  al., 2010). Hypoxia- bial pathogenesis. induced autophagy is also thought to be dependent upon HIF-1 expression of BNIP3 (Maiuri et  al., 2007; Bellot et al., 2009), highlighting the alternative roles of BNIP3 in Acknowledgements the regulation of cell death. However, chronic hypoxia is I am very grateful to Dr Momna Hejmadi for her advice, sup- associated with autophagic cell death via HIF-1 indepen- port and guidance throughout my project. dent activation (Papandreou et  al., 2008; Mazure and Pouysségur, 2010). Funding Under hypoxia, tumour cells can activate other signalling pathways such as the PI3K/AKT (phosphoinositide-3 This study is supported by the University of Bath. kinase) pathway to promote cell survival via growth factors and cytokines (Alvarez-Tejado et al., 2001; Fulda, 2009). In References glioblastoma cells, AKT promoted cell survival by phos- phorylation and inhibition of the pro-apoptotic factor Bad, Alvarez-Tejado, M., Naranjo-Suarez, S., Jimenez, C. et al. 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(2006) Tumor hypoxia and cancer Sullivan, R., Pare, G., Frederiksen, L. et al. (2008) Hypoxia-induced resis- progression, Cancer Letters, 237 (1), 10–21. tance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity, Molecular Cancer Zhou, C., Zhang, X., Liu, F. et al. (2015) Modeling the interplay between Therapeutics, 7 (7), 1961–1973. the HIF-1 and p53 pathways in hypoxia, Scientific Reports, 5, 13–834. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Effect of hypoxia on chemosensitivity to 5-fluorouracil in SH-SY5Y neuroblastoma cells

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BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw005 Research article Eec ff t of hypoxia on chemosensitivity to 5-fluorouracil in SH-SY5Y neuroblastoma cells Hannah Rose Warren* and Momna Hejmadi Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA1 7AY, England *Corresponding author: Email: hannah.r.warren@bath.edu Supervisor: Momna Hejmadi, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA1 7AY, UK. Resistance to chemotherapy is a major obstacle in clinical oncology. Hypoxia is a hallmark of solid tumours as a result of poorly structured tumour neovasculature and is known to be a key contributor to cancer malignancy and reduced drug effectiveness. Hypoxia-inducible factor-1 (HIF-1) mediates the hypoxic response, bringing about adaptive responses to the change in micro- environment. This study investigated the effect of 24 h hypoxia on the sensitivity of SH-SY5Y neuroblastoma cells to 5-fluoro - uracil (5-FU). Cytotoxicity, apoptosis and membrane integrity cell-based assays were carried out to observe changes in cell viability alongside apoptotic and necrotic cell death to determine the molecular mechanisms underlying reduced drug sensi- tivity. Hypoxic cells showed no change in cell viability to 5-FU in comparison to normoxic controls, suggesting that hypoxia conferred reduced drug sensitivity. As a measure of apoptosis, caspase-3/7 levels were significantly higher in hypoxic cells treated with increasing 5-FU concentrations. However, this was associated with less cell necrosis, suggesting that despite increased hypoxia-induced apoptosis, a significant decrease in uncontrolled cell death accounted for the change in viability observed. This finding also implies that alternative cell survival mechanisms could also be activated. Key words: hypoxia, HIF-1, 5-fluorouracil, SH-SY5Y, apoptosis, necrosis Submitted on 7 September 2015; accepted on 6 June 2016 Introduction The hypoxic response is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor. HIF-1 is Hypoxia, a reduction in tissue oxygen levels, is a hallmark of composed of an oxygen-sensitive alpha subunit (HIF-1α) and solid tumour micro-environments and drastically effects a constitutively expressed beta subunit (HIF-1β). Under nor- tumour sensitivity to chemotherapy (Harris, 2002; Tredan moxic conditions, HIF-1α subunits are unstable due to et al., 2007). Regions of hypoxia are associated with distinct hydroxylation of specific proline residues regulated by HIF alterations in the tumour micro-environment, such as increas- prolyl-hydroxylase domain (PHD) proteins, promoting asso- ing acidosis as a response of metabolic switching to glycolysis, ciation with the Von Hippel-Lindau protein (pVHL) ubiquitin reduced nutrient availability and interstitial fluid pressure E3 ligase and subsequent destruction by the proteasome. (Tannock et al., 2002; Brahimi-Horn et al., 2007). Tumours Under hypoxia, HIF-1α is stabilized by the inhibition of PHDs often show a central core of necrotic cells where severe oxygen and instead translocates to the nucleus where it dimerizes with deprivation results in cell death (Brahimi-Horn et al., 2007). HIF-1β. Following recruitment of co-activators, the complex Despite increased angiogenesis, the demand for oxygen always formed binds to the hypoxia response element (HRE) and exceeds the supply, causing hypoxia to remain a constant fea- induces transcription of target genes. The contribution of ture. To survive, tumour cells adapt to these conditions by HIF-1 to drug resistance is complex and has been observed in changes in gene expression, which along with the limited deliv- a range of solid tumours (Sasabe et al., 2007; Liu et al., 2008; ery of drugs to these cells increases resistance to chemotherapy Sullivan et  al., 2008). Active HIF-1 targets ∼1–2% of the and promotes a malignant and more aggressive phenotype. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Research article Bioscience Horizons • Volume 9 2016 human genome, differentially regulating genes involved in a metabolism was shown to confer resistance to RIP-dependent number of biological processes such as angiogenesis, cellular necroptosis via suppression of mitochondrial ROS (Huang metabolism, pH regulation, cell survival and cell death path- et al., 2013). Interestingly, BNIP3 up-regulated under hypoxia ways (Brahimi-Horn et al., 2007). can also mediate a necrosis-like cell death by opening of mito- chondrial pores causing mitochondrial dysfunction independent Cell death and survival pathways under of cytochrome-c release (Vande-Velde et al., 2000). However, this response is thought to be induced only by severe acidosis hypoxia associated with chronic hypoxia (Pouysségur et al., 2006). Hypoxia can induce cell death by a number of distinct path- ways although its effects can be cell type dependent and differ Hypoxia-induced resistance to 5-flurouracil according to severity (Kilic et  al., 2007; Pakravan, 2013). The chemotherapeutic agent 5-fluorouracil (5-FU) is an anti- Severe hypoxia initiates apoptotic or necrotic cell death, metabolite that exerts its cytotoxic effects through the inhibi- whereas under acute milder hypoxia some cells can escape tion of thymidylate synthase and misincorporation into DNA these cell death pathways and survive (Zhou et  al., 2006). inhibiting DNA synthesis (Longley et al., 2003). Mechanisms However, moderate levels of hypoxia can manifest an array of of hypoxia-induced resistance to 5-FU have been reported to responses from an imbalance of cell survival or cell death fac- arise from increased drug efflux expression, G1 phase cycle tors, thus highlighting the inconclusive nature of transient arrest and induced senescence, and inhibition of apoptosis hypoxia within a tumour (Harris, 2002; Cosse and Michiels, (Chang et al., 2006; Sasabe et al., 2007; Yoshiba et al., 2009; 2008; Qiu et al., 2015). Rohwer et al., 2010). However, following DNA damage by Apoptosis is regulated by the members of the Bcl-2 (B-cell chemotherapeutic agents, other cellular responses can be pro- lymphoma 2) protein family which act as positive and negative moted such as autophagy, necrosis and mitotic catastrophe, regulators. Death factors include Bax and Bak which upon acti- suggesting that suppression of a single death pathway may be vation associate with the mitochondrial membrane and cause inadequate to promote survival (Sullivan et al., 2008). This membrane permeabilization releasing cytochrome-c into the study aims to understand how hypoxia influences the sensitiv - cytoplasm. Cytochrome-c binds to Apaf-1 (apoptotic protease ity of SH-SY5Y neuroblastoma cells to 5-fluorouracil, by activating factor 1), which activates caspase 9, in turn cleaving determining changes to the prevalence and type of cell death. caspases 3 and 6. Their activation and cleavage generates a con- trolled signalling cascade permitting cellular changes such as Materials and methods membrane blebbing and programmed disassembly of the cell. Anti-apoptotic proteins such as Bcl-2 and Bcl-xL can shift the All standard reagents were purchased from Fisher Scientific balance to cell survival by inhibiting the function of Bax and unless stated otherwise. Bak. HIF-1α can induce apoptosis by overexpression of pro- apoptotic proteins such as BNIP3 (Bcl-2/adenovirus E1B 19 kDa SH-SY5Y cell line interacting protein 3) and its homologue BNIP3L (NIX) which SH-SY5Y neuroblastoma cells were obtained from American inhibit Bcl-2 and Bcl-xL (Sowter et al., 2001). HIF-1 also pro- Tissue Culture Collection (ATCC) and authenticated using motes p53-dependent apoptosis by increasing the stability of the their testing service. Cells were maintained in Dulbecco’s p53 tumour suppressor gene (Schmid et al., 2004; Zhou et al., Modified Eagle Medium (DMEM-F12 Reduced Serum) 2015). The mutational status of p53 is an important factor as (GIBCO, Invitrogen, Paisley, UK), plus 10% (v/v) fetal bovine loss of functional p53 prevents hypoxia-induced apoptosis serum, 100 µ g penicillin/streptomycin and 0.1 mM l-gluta- (Graeber et al., 1996; An et al., 1998; Sermeus and Michiels, mine. Cells were incubated at 37°C 5% CO and passaged to 2011). Tumour cells can avoid hypoxia-induced apoptosis by 2 maintain optimum confluence for experiments. The cell line up-regulation of anti-apoptotic factors in the Bcl-2 family, along was routinely tested for mycoplasma after 25 passages. with HIF-1-independent pathways such as inhibitor of apopto- sis protein 2 (IAP2) (Dong et al., 2003). Furthermore, in colon Hypoxia exposure cancer cells, the down-regulation of pro-apoptotic factors Bid, Bax and Bad was shown to increase drug resistance, implying Cells were seeded in 96-well plates at a density of 10 cells per protection from apoptosis (Erler et al., 2004). well (in 100 µ l medium) and cultured 24 h prior to use. Concentrations of 5-FU were diluted in culture medium and In contrast, necrosis is characterized by loss of cytoplasmic added to wells. Cells were then incubated under normoxic or integrity, cytoplasmic swelling and nuclear pyknosis, although hypoxic (5% CO , 95% N ) conditions at 37°C for 24 h. 2 2 its biochemical pathways are less well understood. Some necro- sis is regulated termed ‘necroptosis’ by alternative pathways In vitro cytotoxicity by MTT assay involving receptor-interacting protein kinase 1 and 3 (RIP1/ RIP3) which shift cell death from apoptosis to necrosis (Zhang Following exposure to 5-FU, cell viability was determined et al., 2009). Caspase inhibition, ATP availability and mitochon- using an MTT assay. The yellow tetrazolium salt MTT drial generation of reactive oxygen species (ROS) enhance this (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bro- switch (Nikoletopoulou et al., 2013). Under hypoxia, glycolytic mide) is reduced by metabolically active cells by dehydroge- 2 Bioscience Horizons • Volume 9 2016 Research article nase enzymes found in the mitochondria and converted into bated at room temperature and luminescence was measured at an insoluble purple formazan. Isopropanol can be used to dis- time 0, 30 and 60 min after the start of the assay, using a solve the formazan product and its absorbance measured to Promega GloMax plate reader (Promega) at 499 nm (excita- estimate the percentage of viable cells. One milligram per mil- tion) and 521 nm (emission). lilitre of MTT was dissolved in culture medium. Medium was Membrane integrity assay removed from 96-well plates, replaced by 50 µl of MTT solu - tion and incubated for 30 min at 37°C. MTT solution was The CytoTox-ONE™ Homogenous Membrane Integrity removed and the formazan product dissolved in 100 µ l of Assay (Promega) was used to measure the release of lactate isopropanol per well. Absorbance at 560 nm was measured dehydrogenase (LDH) from cells with a damaged membrane using a Modulus Microplate Reader (Turner Biosciences, and was therefore used to indicate the amount of cell necrosis. Promega, Southampton, UK). Cells were plated in 96-well plates as above, with quadruple replicates per plate (normoxic and hypoxic). Following Apoptosis assay hypoxic treatment, 2 µ l of lysis buffer was added to designated The Apo-ONE Homogeneous Caspase-3/7 Assay (Promega) wells containing cells for each treatment plate to act as a max- was used to measure the activity of caspase-3 and −7, the key imum LDH control. Forty microlitres of medium was then effectors of apoptosis. Cells were plated in 96-well plates as transferred to a new 96-well plate (the remaining cells used for before with triplet repeats per plate (normoxic and hypoxic). the Caspase-3/7 assay). Following the manufacturer’s instruc- The protocol was carried out according to the manufacturer’s tions, and working in the dark, one vial of substrate was mixed instructions. Working in the dark, 10 µ l of 100X substrate with 11 ml of buffer. Forty microlitres of assay reagent was was added to 990 µl of buffer. Following hypoxic exposure, a added to each well of transferred medium and incubated at 1:1 ratio using 40 µl of assay reagent was added to each well room temperature for 10 min. Twenty microlitres of stop solu- containing 40 µ l of sample medium. The plates were incu- tion was then added to each well, and the florescence was Figure 1. Effect of hypoxic treatment on cell viability, caspase-3/7 activity and membrane integrity. SHSY-5Y cells were exposed to normoxic and hypoxic conditions for 24 h. (A) Cell viability was measured by MTT assay. Data are expressed as percentage of normoxic controls, and values are represented as means ± SEM from four independent experiments, each containing eight replicates per condition. *Statistical significance using t-test at p < 0.001 (compared with normoxic control). (B) Apoptosis levels were measured by caspase-3/7 activity. Data are expressed as percentages of normoxic controls, and values are presented as means ± SEM from two independent experiments with three replicates per condition at each time interval. *Statistical significance using two-way ANOVA at p < 0.05. (C) Membrane integrity was measured by LDH concentration in the medium. Data are expressed as percentage of maximum normoxic LDH release from lysed cells, and values are presented as means ± SEM from two independent experiments with eight replicates per condition. 3 Research article Bioscience Horizons • Volume 9 2016 Figure 2. Effect of hypoxia and 5-fluorouracil on cell viability, caspase-3/7 activity and membrane integrity. SHSY-5Y cells were exposed to normoxic and hypoxic conditions for 24 h with varying concentrations (0–400 µM) of 5-FU. All statistical tests carried out with two-way ANOVA. (A) Cell viability was measured by MTT assay. Data are expressed as percentages of normoxic no drug control, and values are presented as means ± SEM from three to four independent experiments with eight replicates per treatment. *Statistical significance at p < 0.001. (B) Normoxic apoptosis levels were measured by caspase-3/7 activity taken at time intervals (0, 30 and 60 min after the start of the normoxic assay). Data are expressed as percentage of normoxic no drug controls, and values are presented as means ± SEM from two independent experiments with three replicates per treatment at each time interval. *Statistical significance at p < 0.05 and **at p < 0.001. (C) Apoptosis levels were measured by caspase-3/7 activity taken at 60 min after the start of the assay. Data are expressed as percentage of normoxic control and values represented as means ± SEM from two independent experiments with three replicates per treatment. *Statistical significance at p < 0.05. (D) Membrane integrity was measured by LDH concentration in the medium. Data are expressed as percentage of maximum LDH release from lysed cells and values shown as means ± SEM from two independent experiments with eight replicates per treatment. ® ® measured on a Promega GloMax plate reader (Promega) at were carried out using GraphPad Prism version 6.05 560 nm (excitation) and 590 nm (emission). (GraphPad Software, La Jolla, CA, USA). Statistical analysis Results Data are presented as means ± SEM. Statistical significance was determined using t-tests and one-way/two-way ANOVA The effect of hypoxia and 5-FU on cell viability, apoptosis and with Dunnetts post-hoc as stated. Graphs and statistical tests necrosis was analysed using cell-based assays mentioned above. 4 Bioscience Horizons • Volume 9 2016 Research article Table 1. Hypoxia and 5-FU statistical analysis Initial experiments looked at the effect of hypoxia alone on cell viability, caspase-3/7 activity and membrane integrity. SH-SY5Y cells were incubated in normoxic and hypoxic con- Two-way Source of % of total p-value Significant? ANOVA variation variation ditions for 24 h. Following treatment, there was a significant difference in cell viability between normoxic and hypoxic cells Condition 1.284 0.0124 Yes using the MTT assay (Fig. 1A). Cells treated with hypoxia had 50% reduction in cell viability. Hypoxic data also show a sig- MTT 5-FU 6.086 <0.0001 Yes nificant difference in caspase-3/7 activity compared with nor - Interaction 3.401 0.0057 Yes moxic controls but only after 60 min of assay incubation (Fig.  1B). This suggests that hypoxic cells underwent more Condition 21.68 0.0012 Yes apoptosis than normoxic cells. However, there was no signifi - Caspase cant variation in LDH release between normoxic and hypoxic 5-FU 5.425 0.3926 No 3/7 cells, suggesting no changes in necrosis (Fig. 1C). Interaction 3.074 0.6316 No The effect of 5-FU alone on SH-SY5Y cells was determined Condition 5.586 0.008 Yes using a range of drug concentrations (0–400 µ M) under nor- moxic conditions for 24 h. The high concentration of 5-FU LDH 5-FU 4.259 0.1417 No was determined by previous studies using SH-SY5Y cells and 5-FU (Tieu et al., 1999) as well as Phase II studies of 5-FU in Interaction 1.163 0.679 No other cancer cell lines (Chua et al., 2000; Kim et al., 2003). Results from the MTT assay show that cell viability signifi - cantly decreases with the addition of 5-FU, although this Both normoxic and hypoxic treated cells show a general effect is reduced with increasing concentrations of 5-FU trend of increased LDH release with increasing 5-FU concen- (Fig. 2A). Analysis by two-way ANOVA of caspase-3/7 activ- tration (Fig. 2D), and further analysis by two-way ANOVA ity shows that there is a significant difference in levels of showed that the overall variation between the two conditions apoptosis with the addition of 5-FU compared with the no is significant (Table  1). This supports that hypoxia has drug control (Fig.  2B). However, the level of significance decreased levels of necrotic cell death in comparison. decreases at 400 µ M 5-FU, suggesting that an alternative cell death mechanism could be accounting for the decreased cell viability seen in the MTT assay (Fig. 2A) at this concentra- Discussion tion. Alternatively, the LDH assay shows a trend of increasing Eec ff t of hypoxia and 5-flurouracil on cell LDH release with increasing 5-FU, implying that there is increased necrosis with higher concentrations of 5-FU viability (Fig. 2D). This study shows that SH-SY5Y cells treated with 5-FU under To determine the effect of hypoxia on chemosensitivity, 24 h hypoxia showed reduced sensitivity to 5-FU. As shown SH-SY5Y cells were treated with both varying concentrations by MTT, the addition of 5-FU to oxygenated cells signifi - of 5-FU (0–400 µ M) and 24 h hypoxia. In comparison to cantly reduced cell viability, whereas in hypoxia there was no normoxic conditions, the addition of 5-FU had no significant change in cell viability with increasing 5-FU concentrations. effect on cell viability under hypoxia (Fig. 2A). Furthermore, Similar results of reduced 5-FU sensitivity under hypoxia no significant difference was seen in cell viability between have been previously identified in other cancer cell lines such normoxic and hypoxic cells treated with individual 5-FU as oral squamous cell carcinoma (Yoshiba et al., 2009), gas- concentrations. Statistical analysis using two-way ANOVA tric cancer (Liu et al., 2009) and liver cancer (Yu et al., 2015). revealed the interaction of hypoxia and 5-FU to be signifi - A single 24 h hypoxic treatment was decided upon due to no cant, overall supporting that hypoxia has a significant effect significant variation in HeLa cell viability between normoxic, on reducing the sensitivity of SH-SY5Y cells to 5-FU acute (4 h hypoxia) and intermittent (4 h hypoxia per day for (Table 1). 2 days) hypoxic treatments, and chronic hypoxia (72 h) was toxic to cells (Pool, 2012). However, this 24 h treatment Previous results showed that only after 60 min a signifi - might still not be long enough to see a reduction in cell viabil- cant difference in caspase-3/7 activity could be detected ity using the MTT assay, and therefore, a later time point of between normoxic and hypoxic cells (Fig. 1B). At this same 48 h could also be investigated (Yu et al., 2015). time point, the addition of increasing 5-FU shows a general trend that cells treated with hypoxia had increasing cas- Despite our co-exposure to hypoxia and 5-FU showing a pase-3/7 activity in comparison to normoxic conditions, difference in viability, it would be interesting to see how pre- although this was only statistically significant at 400 µ M exposure to hypoxia, as would be the case in vivo, alters this 5-FU (Fig. 2C). Two-way ANOVA showed that the change in observation. SH-SY5Y cells pre-incubated under hypoxia for condition, rather than 5-FU, has a significant role in trigger - 23 h before drug treatment showed increased viability and a ing apoptosis (Table 1). more resistant phenotype (Hussein et al., 2006). 5 Research article Bioscience Horizons • Volume 9 2016 Figure 3. Hypoxic cell death and cell survival pathways. An overview of different cell responses under varying hypoxia. Hypoxia can promote cell survival by activation of HIF-1-independent pathways such as the PI3K/AKT or Ras/ERK pathways which induce adaptation to the hypoxic micro- environment. ERK can bind to HIF-1α to activate its transcriptional activity. Adaptive responses include up-regulation of angiogenic factors (VEGF), glucose and lactate transporters (GLUT-1, MCT-4), anti-apoptotic factors (Bcl-2, Bcl-XL) and drug efflux pumps (MDR-1) (Brahimi-Horn et al., 2007). Under conditions with low nutrient and ATP availability, hypoxia can inhibit mTOR activity, reducing protein synthesis to conserve energy. Low mTOR activity favours autophagy as a cell survival response which can provide a stress-resistant state. HIF-1 regulation of BNIP3 is involved in activation of autophagy. However, severe hypoxia can induce autophagic cell death. When ATP is conserved, HIF-1 can up-regulate p53, BNIP3 and NIX to promote apoptosis via Bax/Bak activation and subsequent activation of the caspase cascade. Apoptotic cells can undergo secondary necrosis. Necroptosis is an alternative programmed cell death that is independent of caspase activity and is activated by RIP1/RIP3 signalling via death receptors in the absence of caspases (Zhang et al., 2009). When cells are depleted of energy resources under hypoxia, necrosis can be activated by increasing ROS and HIF-1 up-regulation of BNIP3 which causes mitochondrial pore opening. susceptibility to hypoxia-induced apoptosis (Rössler et  al., Eec ff t of hypoxia and 5-flurouracil 2001; Qing et al., 2010). Hypoxia also enhanced the effect of on  apoptosis and necrosis flavopiridol, a cyclin-dependent kinase inhibitor, increasing the percentage of cells undergoing apoptosis compared with Under normoxic conditions, the addition of 5-FU significantly the drug alone (Puppo et al., 2004). Hypoxia-induced apopto- increased the level of apoptosis. At the higher 5-FU, concen- sis in neuroblastoma has been attributed to stabilization of tration of 400 µ M caspase activity decreased which was p53 and caspase activation (Araya et al., 1998; Chen et al., accompanied by increasing LDH release and necrosis. This 2003). The mechanisms accounting for the reduced sensitivity indicates a switch from apoptosis to necrosis with increasing of SH-SY5Y to 5-FU under hypoxia, despite increased apop- 5-FU, suggesting the occurrence of dose-dependent necrosis or tosis, are unclear. It is important to consider that long-term secondary necrosis (Guchelaar et  al., 1998). Hypoxic cells culture increases associated chromosomal abnormalities and showed higher levels of apoptosis after 60 min which that N-myc overexpressing populations could result in pre- increased with the addition of 5-FU, and at 400 µM, this was dominant apoptosis under hypoxia, while cells with normal significantly higher than normoxic cells. This was associated N-myc survive (Rössler et al., 2001; Ushmorov et al., 2008; with reduced overall LDH release, implying that there is less Poljaková et al., 2014). necrosis and more apoptosis. Interestingly, in a previous report, acute (4 h) and intermit- Similar results were reported in other neuroblastoma cell tent (4 h per day for 2 days) hypoxia showed significantly lines where amplification of the N-myc oncogene increased 6 Bioscience Horizons • Volume 9 2016 Research article lower levels of apoptosis in HeLa cells compared with nor- Conclusion moxic conditions (Pool, 2012). Our 24 h hypoxia is more severe and sustained, highlighting the differential role of vary- This study has shown that hypoxia reduces sensitivity to 5-FU ing hypoxia on cell death mechanisms. Neuroblastoma cells in a neuroblastoma cell line. Despite increased apoptosis, cell are also inherently different in terms of gene expression and viability remained unchanged with increasing 5-FU concen- sensitivity to hypoxia due to different rates of cell growth and trations, suggesting that along with decreased necrosis other cell death (Jögi et al., 2004). cell survival mechanisms must be in place. Further work is needed to confirm the exact mechanisms involved in reduced Alternatively, hypoxia can trigger other survival mecha- drug sensitivity of SH-SY5Y cells under hypoxia. To provide a nisms, which alongside decreased necrotic cell death, must comprehensive view of chemoresistance under hypoxia, other play a role in maintaining the cell viability and reduced drug mechanisms of cell death and survival beside apoptosis and sensitivity observed. Hypoxia can promote cellular adapta- necrosis should be investigated. Autophagy is known to have tion and survival by suppression of protein synthesis and a role during hypoxia, and assays would provide an insight cell growth via mTOR (mammalian target of rapamycin) into its adaptive or detrimental role under hypoxia. (Arsham et al., 2003; Pouysségur et al., 2006). Low mTOR activity under hypoxia favours the activation of autophagy, a survival response to remove damaged or unwanted organ- Author’s biography elles to preserve cell fitness and oxygen homeostasis ( Zhang H.R.W. graduated from the University of Bath in 2015 with a et  al., 2008). Here autophagy is considered protective first class honours degree in BSc Molecular and Cellular against apoptosis and can provide a stress-resistant state Biology. This research was carried out as part of her final-year supporting chemotherapeutic resistance (Chen et al., 2010). dissertation project and was chosen due to her interest in the Functional inhibition of autophagy in colon cancer cells was mechanisms of cancer drug resistance. Other fields of interest shown to restore chemosensitivity to 5-fluorouracil and include clinical pathology, infection and immunity and micro- increase tumour cell death (Li et  al., 2010). Hypoxia- bial pathogenesis. induced autophagy is also thought to be dependent upon HIF-1 expression of BNIP3 (Maiuri et  al., 2007; Bellot et al., 2009), highlighting the alternative roles of BNIP3 in Acknowledgements the regulation of cell death. However, chronic hypoxia is I am very grateful to Dr Momna Hejmadi for her advice, sup- associated with autophagic cell death via HIF-1 indepen- port and guidance throughout my project. dent activation (Papandreou et  al., 2008; Mazure and Pouysségur, 2010). Funding Under hypoxia, tumour cells can activate other signalling pathways such as the PI3K/AKT (phosphoinositide-3 This study is supported by the University of Bath. kinase) pathway to promote cell survival via growth factors and cytokines (Alvarez-Tejado et al., 2001; Fulda, 2009). In References glioblastoma cells, AKT promoted cell survival by phos- phorylation and inhibition of the pro-apoptotic factor Bad, Alvarez-Tejado, M., Naranjo-Suarez, S., Jimenez, C. et al. 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