Doctoral Degrees (Physiological Sciences)
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Browsing Doctoral Degrees (Physiological Sciences) by browse.metadata.advisor "Loos, Benjamin"
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- ItemAnthracycline-induced cardiotoxicity : the role of proteolytic pathways(Stellenbosch : Stellenbosch University, 2012-03) Sishi, Balindiwe J. N. (Balindiwe Jennifer Nonkosazana); Engelbrecht, Anna-Mart; Loos, Benjamin; Van Rooyen, Jacques; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR) are two of the most effective drugs known for the treatment of systemic neoplasms and solid tumours. However, their clinical use is often hampered by their dosedependent cumulative cardiotoxicity, which leads to irreversible and fatal druginduced congestive heart failure. The mechanism by which ACs induces heart damage is not fully understood. Recent reports have indicated that DXR activates autophagy and ubiquitin proteasome-mediated degradation of specific transcription factors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases, MuRF-1 and MAFbx. The aim of the first part of the study was therefore to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate the signalling mechanisms involved. Although this model was ideal in allowing the investigation of the signalling pathways which are affected by DNR, it did not allow for further exploration or manipulation of signalling pathways that may be of potential benefit in this context. The in vitro model was therefore used to validate the hypothesis that increased autophagy alleviates AC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims for the second part of the study were (i) to characterize the effect of DXR in H9C2 cells, (ii) to determine whether the induction/inhibition of autophagy in combination with DXR alleviates cytotoxicity and (iii) to investigate the influence of increased/decreased autophagy in combination with DXR on reactive oxygen species (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stress and the ubiquitin proteasome pathway. In the final part of this study, an in vivo model was used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumour bearing mouse model of acute DXR-induced cardiotoxicity. Material and Methods: Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped for biochemical analysis. H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy) for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24 hrs or a combination of these drugs. Following treatment, cells were harvested and assessed for cell death, proteolytic activity and oxidative stress using western blotting, fluorescence microscopy and flow cytometry. In the final phase of the study, twenty-four female mice were injected at 8 weeks with a mouse breast cancer cell line (EO771) and after observation of tumour growth, animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), two injections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After the experimental protocol, mice were terminated and their hearts were rapidly excised. The hearts were divided cross-sectionally and utilized for biochemical and histological analyses. Results and Discussion: DNR treatment significantly attenuated myocardial function and increased apoptosis in the ex vivo heart model. DNR-induced cardiac cytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1 and MAFbx as well as a significant increase in two markers of autophagy, beclin-1 and LC-3. These changes observed in the heart were also associated with attenuation of the PI3-kinase/Akt signalling pathway. The augmentation of autophagy with rapamycin before DXR treatment significantly reduced cell death in the in vitro model. Indeed, rapamycin treatment demonstrated to be a vital survival mechanism for acute DXR-induced cardiotoxicity as it decreased cellular ROS production, improved mitochondrial function and prevented nuclear translocation of DXR. Moreover, these changes in cardiomyocytes were also associated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model was developed to confirm the results obtained in the in vitro study. It was demonstrated that acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibition of autophagy and increased proteolysis via the UPP. These findings were associated with a reduction in body weight and cardiomyocyte cross-sectional area. The cardiotoxic effects of DXR were substantially reduced when autophagy was induced with rapamycin. Taken together, our data strongly indicates that it is possible to attenuate the cardiotoxic effects of doxorubicin in cancer patients by carefully controlling the levels of autophagy using rapamycin as adjuvant therapy.
- ItemThe effects of nutrient deprivation on macroautophagic flux and chaperone-mediated autophagy in a model of alzheimer's disease(Stellenbosch : Stellenbosch University, 2018-12) Ntsapi, Matlakala Claudia; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: Alzheimer’s disease (AD) is a devastating neurodegenerative disease characterized by progressive cognitive impairment, particularly in brain regions crucial for learning and memory. These symptoms are caused by neuronal death resulting from two pathological features: extracellular senile plaques composed of aggregated amyloid-beta (Aβ) peptides, and intracellular neurofibrillary tangles generated by the hyperphosphorylation of tau protein. Although AD is a multifactorial disease, much of the AD research continues to be guided by the amyloid cascade hypothesis, which posits that Aβ aggregation is the key initiate in AD pathogenesis. Aβ is generated from the proteolytic cleavage of the amyloid precursor protein (APP) by β - and γ-secretase. Accordingly, research efforts to modulate APP processing, and better clarify the mechanisms that regulate intracellular Aβ metabolism and clearance during AD progression have been explored in the treatment of AD. Autophagy, a lysosome-based proteolytic pathway that plays a crucial role in intracellular protein quality control, has been implicated in both the production and clearance of Aβ peptide. Cumulative evidence shows that AD-related autophagic dysfunction coincides with the detection of Aβ within autophagic vacuoles (AVs) that accumulate within dystrophic neurites with the initial increase in Aβ neurotoxicity. Therefore, autophagy dysfunction may exacerbate Aβ pathology and further augment disease progression; however, when in this context autophagy becomes dysfunctional remains unclear. Moreover, although it is known that Aβ levels themselves may induce autophagy, how long autophagy remains upregulated and functional in this process is unclear. It also remains unclear whether autophagy plays a causative, or protective role in Aβ neurotoxicity; or whether autophagy dysfunction is a consequence of the disease process itself. Therefore, the aims of this study were (i) to characterize the expression profile of key amyloidogenic pathway proteins, both macroautophagy and chaperone-mediated autophagy (CMA) proteins as well as the extent of neuronal toxicity using a unique APP overexpression model, (ii) to dissect the interplay between proteolytic pathways and cell death markers in the context of APP overexpression using a proteomics approach, (iii) to assess macroautophagic flux in the context of APP overexpression and to unravel the extent of autophagy dysfunction, (iv) to assess the contribution of macroautophagy and CMA in Aβ clearance and neuronal toxicity by modulating each pathway, and (v) to assess the effects of prolonged intermittent fasting (IF) on the modulation of macroautophagy and CMA in a paraquat (PQ)-induced in vivo brain injury model. Methods: A unique AD overexpression model, the N2a mouse neuroblastoma cell line stably overexpressing the human Swedish double mutation was utilized. APP overexpression was characterized, and the induction of macroautophagy, CMA, and apoptosis was assessed over time using a combination of cell viability assays, western blot analysis, fluorescence microscopy, transmission electron microscopy (TEM), and correlative light and electron microscopy techniques. Moreover, the effect of APP overexpression on a global proteome level was quantified using high resolution liquid-chromatography coupled to tandem mass spectrometry. Finally, a PQ – induced brain injury model was established and utilized to assess the effects of prolonged IF on macroautophagy and CMA using GFP–LC3 transgenic mice. Mice were injected twice weekly with 10 mg/kg PQ for a duration of 3 weeks. A prolonged IF protocol of 48 hrs fasting, followed by 24 hrs refeeding was implemented for a duration of 3 weeks. Modulation of macroautophagy and CMA following chronic oxidative stress exposure, and prolonged IF was evaluated in selected brain regions by western blot analysis, fluorescence microscopy, comparative haematoxylin and eosin staining, and TEM analysis. Results: The results indicate that APP overexpression leads to prominent apoptosis induction after 48 hrs and activates the autophagy machinery in a time-dependent manner. To our surprise, macroautophagic flux analysis reveals that autophagy is upregulated upon APP overexpression but remains elevated in the presence of apoptosis induction. Our CMA analysis indicates that APP overexpression activates the CMA machinery, particularly during the 48 hrs time point. However, induction of apoptosis proceeded despite elevated levels of CMA activity. Next, our proteome analysis reveals a time-dependent increase in APP proteinprotein interaction partners over time. Cumulatively, the in vitro results suggest that the modulation of macroautophagy and CMA augments Aβ clearance and mitigates neuronal toxicity. In vivo, a significant decrease in cytochrome c, and 4HNE expression were observed with prolonged IF intervention in selective brain regions. These changes were associated with elevated levels of macroautophagy and CMA induction, as evidenced by the significant increase in LC3II and LAMP2A protein expression. Therefore, suggesting that protection was brought about by the prolonged IF intervention through the modulation of macroautophagy and CMA. Conclusion: Our findings indicate that autophagy is upregulated in the presence of high levels of APP and Aβ, and to our surprise, remains upregulated even in the presence of apoptosis induction, suggesting an insufficient autophagy response in the mitigation of Aβ neurotoxicity. However, enhanced Aβ clearance was observed with a sufficiently high autophagy response even during 48 hrs APP overexpression, suggesting that autophagy modulation may be a viable treatment approach long into disease progression. These findings were also confirmed with prolonged IF intervention, where markers of apoptosis, and lipid peroxidation were notably decreased in brain regions associated with neurodegeneration. Further studies, specifically using in vivo APP overexpression models are warranted to further verify the clinical use of autophagy control.
- ItemAn evaluation of the hepatic proteomic signature in identifying cancer tolerance and resistance mechanisms in a mouse allograft system(Stellenbosch : Stellenbosch University, 2017-03) Van Niekerk, Gustav; Engelbrecht, Anna-Mart; Loos, Benjamin; Nell, Theo A.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background The unfavourable therapeutic index of most treatment modalities has greatly impeded progress in the development of effective cancer therapy. Therefore a need exists for treatment modalities that are less taxing on a patient’s health status (i.e. maintain a patients reserve capacity and thus prolong survival) while additionally not invoking counter evolutionary strategies from tumour cells. Plant biologist have long distinguished between the host’s ability to accommodate pathogen burden, as oppose to its ability to antagonise pathogen load. Hence, the disease trajectory is not only dependent on the hosts’ ability to resist an infection, but also on the capacity to tolerate pathogen burden. This distinction have only recently been applied to animals. A number of observations suggest that this distinction may be of great immunological relevance, including the prevalence of asymptomatic carriers and natural variation in the population with regards to disease progression. Thus, the tolerance/resistance (T/R) paradigm represents a novel approach for understanding disease progression. We hypothesise that similar mechanisms might underlie host-tumour dynamics. Study aims and experimental design The current study aimed to evaluate the application of the T/R framework within an oncological context. A syngeneic mice model system was used to compare tolerance and resistance between two cancer cell lines. C57BL/6 mice were inoculated with either mammary carcinoma cells (EO771) or melanoma cells (B16). In a clinical setting, health status would not only be influenced by tumour load, but also by therapeutic interventions such as cytotoxic therapies, which must also be tolerated. Thus, a second study was performed using chemotherapeutic regimes as a variable to explore the effect of high (5 mg/kg) and low (2 mg/kg) dose doxorubicin (DXR) treatment on tolerance and resistance in mice. In addition, attempts were made to identify mechanisms underlying differences between groups with regards to variation in tolerance and resistance. To this end, a combination of immunoblotting and proteomic analyses were performed. Methodology: quantifying tolerance and resistance Resistance was quantified as the slope of a regression line, with tumour volume as response variable, and time as independent variable. Tolerance was measured similarly, but with body weight as response variable and tumour load as independent variable. Differences in regression slopes was used to compare tolerance and resistance. To confirm tolerance, differences in gastrocnemius muscle cross-sectional area (MCA) were compared between groups. Results Mice inoculated with melanoma (B16) cells showed a significantly lower resistance compared to mice inoculated with breast cancer EO771 cells. With regards to tolerance, B16 cells also exhibited lower tolerance, though tests for homogeneity of regression slopes demonstrated that these differences did not reach significance (p = 0.0856). Similarly, B16 and EO771 groups did not exhibit any difference in MCA. Comparing the effect of high and low dose DXR on mice bearing EO771 revealed that DXR decreases resistance: both low dose and higher dose DXR increased tumour growth as demonstrated by significantly steeper slopes in DXR groups compared to the tumour control group. In order to explain the increase in EO771 tumour growth in mice receiving DXR, the activation of a panel of signalling proteins associated with cell growth and survival (cRaf, ERK, p38 MAPK, JNK, PTEN, PI3Kp85, PDK1, Akt, mTOR, Bcl-2) as well as apoptotic markers (Caspase 3, 8 and 9) in tumour samples were evaluated by western blot analyses. However, the only significant finding include elevated ERK activation in mice receiving DXR, suggesting that extracellular signalling molecules might drive tumour growth. Since the liver plays a critical role in energy homeostasis, as well as in the production and clearance of circulating factors, western blot analyses were performed on liver samples. Markers of autophagy (p62 and LC3B-II) as well as growth signalling proteins (Akt and mTOR) and apoptosis (Caspase 3) were evaluated by western blot analyses. Mice inoculated with B16 demonstrated a marked increase in both p62 and LC3B-II, signifying an increase in autophagosome pool size, most likely due to dysfunctional lysosomal fusion. Surprisingly, other makers in both EO771 and B16 did not significantly differ from control liver samples. Subsequently, liver proteomics were performed making use of a Gene Ontology approach in order to describe biological, functional, structural and other processes that are uniquely altered between groups. Interestingly, a comparison between livers of mice inoculated with B16 melanoma cells and EO771 breast cancer cells also suggested that autophagic activity was not upregulated compared to the control group. DXR groups also did not exhibit differences in autophagic processes, though proteins involved in the proteasomal pathway were upregulated in mice receiving high doses of DXR. An increase expression of enzymes associated with retinoic acid metabolism was observed in the B16 group, which might explain decrease tolerance and resistance in this group. An increase in steroid metabolism was also observed in mice receiving DXR. Since cholesterol form a key component of cell membranes, it is possible that cholesterol synthesis might enable rapidly growing tumours of mice receiving DXR. Finally, concurrent up- and downregulation of certain proteins involved in radical scavenging in DXR mice might suggest a differential free radical scavenging response, thus explaining why anti-oxidant therapies have not proven successful in clinical settings in response to DXR. Collectively, these observations highlight alteration in hepatic activities through which tolerance and resistance mechanism might manifest. In summary, this study have demonstrated the implementation of the T/R framework within an oncological setting. Evidence suggest that defects in hepatic autophagy might contribute to lower tolerance, and possibly also resistance. Autophagy was not significantly upregulated in response to DXR which was associated with lower tolerance. Similar, mice inoculated with B16 tumours exhibited lower tolerance as well as evidence for suppressed lysosomal fusion with autophagosome. These observations suggest that a compromised autophagic apparatus might contribute towards the lower tolerance. Proteomic results are also suggestive of a potential role played by altered liver metabolism, including retinoic acid and steroid metabolism. Future studies evaluating the role of this pathways might identify novel tolerance-promoting pathways.
- ItemManipulation of the autophagic pathway sensitises cervical cancer cells to cisplatin treatment(Stellenbosch : Stellenbosch University, 2013-03) Leisching, Gina Renata; Engelbrecht, Anna-Mart; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction Cisplatin has been widely used to treat solid tumours and much success has come from the use of this drug in the treatment of head and neck, ovarian, testicular, cervical and small-cell lung cancers. However, the success of cisplatin treatment is limited due to its dose-limiting toxicity and its resulting side-effects, such as nephro- and ototoxicity. The devastating side-effects induced by cisplatin treatment provided the platform for this study whereby the aim was to lower the concentration of cisplatin while maintaining its cancer-specific cytotoxic action. Equally concerning is, cisplatin resistance which is becoming increasingly common, and this radically limits the clinical efficacy and utility of the drug. Adjuvant therapy has thus become necessary in an attempt to possibly curb or lessen the extent of cisplatin resistance. Due to the large body of evidence implicating the importance of autophagy in cancer, the prospect of targeting this mechanism has generally been accepted. Various chemotherapy agents induce autophagy in cancer cells; however the effect of cisplatin on autophagic induction has not been very well explored. We thus hypothesise that the manipulation of the autophagic pathway will sensitise cancer cells to a low concentration of cisplatin treatment. Furthermore, due to the functional interaction between Bcl-2 and Beclin-1 and its role in the regulation of autophagy, ratio analysis of Beclin-1 to Bcl-2 as means of detecting the role of autophagy within the cell under homeostatic and treatment/stress conditions has been conducted. Additionally, Bcl-2 has a prominent role in the malignant cell and it’s over-expression has been found to confer resistance in a variety of cancerous cell lines. We therefore hypothesise that the silencing of Bcl-2 prior to cisplatin treatment will sensitise cervical cancer cells to apoptosis and increase the Beclin-1/Bcl-2 ratio in favour of apoptosis. Materials and Methods Three human cervical cell lines were used: a non-cancerous ectocervical epithelial cell line (Ect1/E6E7) and two cancerous cervical cell lines (HeLa and CaSki). In order to determine a concentration of cisplatin that was non-toxic to the non-cancerous Ect1/E6E7 cell line, a dose-response was performed. With the use of an autophagy inhibitor (bafilomycin A1) and an autophagy inducer (rapamycin), autophagic flux capacities were assessed in each cell line through the Western blotting technique. In order to assess whether the chosen concentration of cisplatin induced autophagy, flow cytometry with the use of a Lysotracker™ dye was utilised, as well as analysis of autophagy protein levels (LC-3 II, Beclin-1 and p62). Autophagy modulation was achieved through two methods: pharmacological modulation with use of two recognised agents, namely bafilomycin A1 and rapamycin, and biological manipulation with the use of ATG5 and mTOR mRNA silencing. The effects of different treatment regimes on cell death was assessed with the use of PARP and caspase-3 cleavage through Western blotting, caspase-3/-7 activity (Caspase-Glo®), PI inclusion, LDH release and MTT reductive capacity. Additionally the effects of these treatment regimes on cell-cycle progression were also analysed. Beclin-1 and Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin in HeLa and CaSki cells. To assess the reliance of the cervical cancer cells on Bcl-2 after cisplatin treatment, Bcl-2 knock-down was achieved through RNA interference, where after the Beclin-1/Bcl-2 ratio was assessed as well as apoptosis with the use of cleaved PARP analysis (Western blotting) and Caspase-Glo©. For the ex vivo analysis, biopsies were collected from patients undergoing routine colposcopy screenings and hysterectomies at Tygerberg Hospital, Tygerberg, Western Cape. A total of 10 normal, 29 low-grade squamous intraepithelial lesions (LSIL), 33 high-grade squamous intraepithelial lesions (HSIL) and 13 carcinoma biopsies were collected for analysis, where after the expression profiles of two autophagy markers (mTOR and LC-3 II), as well as one anti-apoptotic marker (Bcl-2) were assessed. Protein levels were analysed through Western blot and confirmed through immunohistochemistry. Results Dose-response curves revealed that 15 μM of cisplatin did not induce cell death in the normal cervical epithelial cell line (Ect1/E6E7) and was therefore utilised through-out the remainder of the study. It was additionally determined that the CaSki cells were more resistant to cisplatin treatment when compared to the HeLa and Ect1/E6E7 cells. Autophagic flux analysis revealed that, although all three cell lines were cervix derived, their autophagic flux capacities differed. It was observed that the chosen concentration of cisplatin was able to induce autophagy in all three cell lines, with the HeLa cells demonstrating a particularly pronounced response. Autophagy modulation in conjunction with cisplatin treatment revealed the following: Autophagy inhibition with bafilomycin A1 lead to significant increases in caspase-3 and PARP cleavage and LDH release in both cervical cancer cell lines. The inhibition of autophagy through silencing of ATG5 induced caspase-3 cleavage and agrees with results obtained from pharmacological inhibition of autophagy with bafilomycin A1. In addition to autophagic induction, a low concentration of cisplatin induced the up-regulation of Bcl-2, which when silenced significantly improved cisplatin-induced apoptosis in both cervical cancer cell lines. Analysis of the expression profiles of mTOR and LC-3 in normal, pre-malignant (LSIL and HSIL) and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma of the cervix. Additionally, Bcl-2 expression is significantly increased in cervical carcinoma tissue, which agrees with results from other studies. Conclusion Autophagic flux capacities between the three cell lines investigated, derived from the same organ, differ significantly. This should be taken into consideration when autophagic modulation is being used as an adjuvant treatment. With regard to chemotherapy treatment in cervical cells, a low-concentration of cisplatin significantly induces autophagy in malignant and non-malignant cervix-derived cell lines where it serves a pro-survival mechanism. Inhibition of autophagy with bafilomycin A1 and ATG5 siRNA confirmed this survival effect in both cancerous cell lines where apoptosis was significantly increased. Interestingly, rapamycin pre-treatment together with cisplatin did not induce significant levels of apoptosis in HeLa cells where autophagy induction may have provided additional protection from the cytotoxic effects of cisplatin. Therefore the inhibition of autophagy through pharmacological and biological inhibition improves the cytotoxicity of a low concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer. Bcl-2 up-regulation in response to cisplatin treatment also serves as a protective mechanism by which cervical cancer cells survive. The extent of apoptotic cell death observed after biological inhibition of Bcl-2 reiterates the fact that this response may be exploited in order to favour the use of lower concentrations of cisplatin. Analysis of clinical specimens emphasised the value of the in vitro work: Cervical cancer biopsies had increased expression of both LC-3 II and Bcl-2, indicating autophagy induction and apoptosis inhibition, respectively. Thus two novel methods of improving cisplatin cytotoxicity have been demonstrated in the following study. Treatment regimens may administer more frequently and prolonged due to the minimal side-effects that accompanies low-dose cisplatin treatment.
- ItemThe role of the AHNAK protein in breast cancer : implications for tumour metastasis and chemoresistance(Stellenbosch : Stellenbosch University, 2016-12) Davis, Tanja Andrea; Engelbrecht, Anna-Mart; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction – Cancer continues to have a significant impact on society. While there has been much success in characterising tumours and identifying targetable markers, two major problems are still faced today, namely therapeutic failure and advanced progression of the disease. The human AHNAK protein is a giant scaffold protein involved in multiple cellular processes and has now also been suggested to be associated with cancer, particularly with regards to tumour metastasis and chemoresponse. However, limited information and several contradicting findings have contributed to a poor understanding of the role of AHNAK in cancer. Thus, we aimed to characterise the AHNAK protein in cancer by determining the role of the protein in the chemotherapeutic response of breast cancer to doxorubicin (DXR) and also in cellular migration. Methods – For the in vitro model the non-metastatic DXR-sensitive epithelial-like MCF-7 and metastatic DXR-resistant mesenchymal-like MDA-MB-231 cell lines were used. We performed DXR treatments and assessed AHNAK’s protein expression and intracellular localisation. We also assessed these properties in a tumour-bearing mouse model. AHNAK knockdown and overexpression was achieved by means of transient plasmid transfections in both cell lines and following DXR treatments we assessed apoptotic marker expression, cell cycle modulation, epithelial-mesenchymal transition (EMT) marker expression and cellular migration. Results – DXR induced dose-independent and dose-dependent changes in AHNAK protein expression in MCF-7 and MDA-MB-231 cells, respectively, but it did not affect its intracellular localisation in these cells. In the tumour-bearing mouse model DXR also induced dose-dependent changes in AHNAK expression without affecting its localisation, similar to the MDA-MB-231 cells. In the MDA-MB-231 cells, DXR promoted apoptosis inhibition by decreasing cPARP and cCasp7 expression. Knockdown of AHNAK prevented this inhibition while overexpression induced a similar inhibitory effect. With cell cycle analyses we observed that DXR also resulted in S phase arrest in these cells. AHNAK knockdown completely prevented the DXR-induced cell cycle arrest while overexpression was sufficient to cause such an arrest on its own. No significant effects were observed with these experiments in the MCF-7 cells. DXR induced EMT in the MCF-7 cells but AHNAK knockdown or overexpression did not affect this. In MDA-MB-231 cells DXR treatment showed a trend of decreased EMT and while AHNAK knockdown had no effect on this, its overexpression showed clearer evidence of EMT reduction. AHNAK knockdown also had no major effects on cell migration in both cell lines, although its overexpression generally decreased cellular migration. Conclusions – We show that AHNAK plays a novel role in the DXR-response of breast cancer cells and this involved AHNAK’s expression, apoptosis inhibition and cell cycle modulation. Possible molecular mechanisms are proposed but require further investigation. Our results regarding the role of AHNAK in tumour cell migration is less clear and contradicting when compared to other studies. These results may have potential therapeutic implications with regards to the modulation of DXR response to improve treatment efficacy.