Academic literature on the topic 'Autophagy, breast cancer, IKBKE'

IKBKE is an oncogene in triple-negative breast cancer (TNBC), and we demonstrate that IKBKE small interfering RNA (siRNA) inhibits the proliferation, migration, and invasion of TNBC cells. Despite the recent success of siRNA therapeutics targeting to the liver, there still remains a great challenge to deliver siRNAs to solid tumors. Here, we report a hybrid nanocomplex to co-deliver the IKBKE siRNA and cabazitaxel to TNBC to achieve an optimal antitumor effect. The nanocomplex is modified with hyaluronic acid to target CD44 on TNBC cells. The nanocomplex shows higher cellular uptake and better tumor penetration of the encapsulated cargos. The nanocomplex also exhibits high tumor accumulation and antitumor activity in an orthotopic TNBC mouse model. Encapsulation of cabazitaxel in the nanocomplex enhances the activity of the IKBKE siRNA. The hybrid nanocomplex provides a novel and versatile platform for combination therapies using siRNAs and chemotherapy.

Breast cancer is a heterogeneous disease consisting of different biological subtypes, with differences in terms of incidence, response to diverse treatments, risk of disease progression, and sites of metastases. In the last years, several molecular targets have emerged and new drugs, targeting PI3K/Akt/mTOR and cyclinD/CDK/pRb pathways and tumor microenvironment have been integrated into clinical practice. However, it is clear now that breast cancer is able to develop resistance to these drugs and the identification of the underlying molecular mechanisms is paramount to drive further drug development. Autophagy is a highly conserved homeostatic process that can be activated in response to antineoplastic agents as a cytoprotective mechanism. Inhibition of autophagy could enhance tumor cell death by diverse anti-cancer therapies, representing an attractive approach to control mechanisms of drug resistance. In this manuscript, we present a review of autophagy focusing on its interplay with targeted drugs used for breast cancer treatment.

Fibrous sheath interacting protein 1 (FSIP1) is a cancer antigen expressed in the majority of breast cancer tissues and is associated with poor prognosis. However, the role of FSIP1 in the progression and drug sensitivity of triple-negative breast cancer (TNBC) has not been explored. Here, we show that FSIP1 deficiency by shRNA-mediated knockdown or CRISPR-Cas9–mediated knockout significantly inhibits the proliferation and invasion of TNBC cells and impairs chemotherapy-induced growth inhibition in vivo. Computational modeling predicted that FSIP1 binds to ULK1, and this was established by coimmunoprecipitation. FSIP1 deficiency promoted autophagy, enhanced AMP-activated protein kinase (AMPK) signaling, and decreased mechanistic target of rapamycin (mTOR) and Wnt/β-catenin activity. In contrast, knockdown of AMPK or inhibition of autophagy restored the sensitivity to chemotherapy drugs in TNBC cells. Our findings uncover a role of FSIP1 as well as mechanisms underlying FSIP1 action in drug sensitivity and may, therefore, aid in design of TNBC therapies.

Autophagy is one of the major responses to stress in eukaryotic cells and is implicated in several pathological conditions such as infections, neurodegenerative diseases and cancer. Interestingly, cancer cells take full advantage of autophagy both to support tumor growth in adverse microenvironments and to oppose damages induced by anti-neoplastic therapies. Importantly, different human oncogenes are able to modulate this survival mechanism to support the transformation process, ultimately leading to “autophagy addiction”. Still, oncogenic signaling events, impinging on the control of autophagy, are poorly characterized, limiting our possibilities to take advantage of these mechanisms for therapeutic purposes. Here, we screened a library of activated kinases for their ability to stimulate autophagy. By this approach, we identified novel potential regulators of the autophagic process and, among them, the IKBKE oncogene. Specifically, we demonstrate that this oncoprotein is able to stimulate autophagy when overexpressed, an event frequently found in breast tumors, and that its activity is strictly required for breast cancer cells to support the autophagic process. Interestingly, different oncogenic pathways typically involved in breast cancer, namely ERBB2 and PI3K-AKT-MTOR, also rely on IKBKE to control this process. Ultimately, we show that IKBKE-dependent autophagy is necessary for breast cancer cell proliferation, suggesting an important supporting role for this oncogene and autophagy in these tumors.

Aquest és el primer informe que demostra que l'autofàgia està mecànicament vinculat al manteniment de les cèl•lules tumorals que expressen alts nivells de CD44 i baixos nivells de CD24, que són típics de les cèl•lules mare del càncer de mama. Els nostres resultats actuals proporcionen una nova visió de com la divisió mitocondrial s'integra a la xarxa de la transcripció impulsada per factors de reprogramació, especifica de la pluripotència única de les cèl•lules mare. L'autofàgia pot controlar la refractarietat de novo de carcinomes de mama amb el gen HER2 amplificat per l'anticòs monoclonal trastuzumab (Herceptin). Per tant, el tractament de combinació amb trastuzumab i cloroquina, com a fàrmac anti-malàric i inhibidor de l’autofàgia, suprimeix radicalment el creixement del tumor en un xenoempelt de tumor completament refractari a trastuzumab en un model murí. L’addició de cloroquina amb els règims amb trastuzumab pot, per tant, millorar els resultats en les dones amb càncer de mama HER2. Aquesta és una àrea molt emocionant i molt prometedora de la investigació del càncer, com la modulació farmacològica de l'autofàgia sembla augmentar l'eficàcia dels règims contra el càncer disponibles en l'actualitat i s'obre el camí per al desenvolupament de noves estratègies terapèutiques combinatòries que s'espera que contribueixin a l'eradicació del càncer.
This is the first report demonstrating that autophagy is mechanistically linked to the maintenance of tumor cells expressing high levels of CD44 and low levels of CD24, which are typical of breast cancer stem cells. Our current findings provide new insight into how mitochondrial division is integrated into the reprogramming of the factors-driven transcriptional network that specifies the unique pluripotency of stem cells. Autophagy may control the de novo refractoriness of HER2 gene-amplified breast carcinomas to the monoclonal antibody trastuzumab (Herceptin). Accordingly, treatment with trastuzumab and chloroquine, as antimalarial drug and inhibitor of autophagy, radically suppresses tumor growth in a tumor xenograft completely refractory to trastuzumab in a mouse model. Adding chloroquine to trastuzumab-based regimens may therefore improve outcomes among women with autophagy-addicted HER2-positive breast cancer. This is a very exciting and highly promising area of cancer research, as pharmacologic modulation of autophagy appears to augment the efficacy of currently available anticancer regimens and opens the way to the development of new combinatorial therapeutic strategies that will hopefully contribute to cancer eradication.

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D3 also enhanced radiosensitivity and promoted autophagy in MCF7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D3 was not attenuated by either a pharmacological or genetic block to autophagy; this was due largely to the promotion of apoptosis via the suppression of protective autophagy that occurs in response to radiation alone. Moreover, pharmacological blockade of autophagy did not sensitize noncancerous MCF10a cells to radiation; conversely, 4T1 mouse mammary tumors were highly sensitive to pharmacological inhibition of autophagy, suggesting selective radiosensitization against cancer cell lines. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D3). In addition, this work highlights the technical challenge of establishing the potential cytotoxic function of autophagy in an experimental system where the cytoprotective function may be concurrently expressed.

During severe hypoxia (<0.01% oxygen) the protein folding machinery becomes dysfunctional, resulting in the accumulation of unfolded proteins with consequent endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) and autophagy, a process involved in the physiological turnover of cytoplasmic components. The link between the UPR and autophagy is not clearly defined. The aim of this thesis is to investigate the role of the induction of UPR under severe hypoxia in tumour survival and resistance to therapy. The results of this research suggest that the activating transcription factor 4 (ATF4), a component of the PKR-like ER kinase (PERK) pathway, fundamental in the UPR, is required for the ER-stress induced upregulation of autophagy. Mechanisms other than hypoxia for UPR induction were investigated, using the proteasome inhibitor bortezomib (BZ). BZ treatment increased ATF4 protein levels in MCF7 cells, even transfected with short-interference RNA (siRNA) against the classical UPR activator PERK, suggesting that the proteasomal stabilization is likely the main mechanism for ATF4 protein accumulation. The induction of autophagy by BZ is dependent upon the upregulation of the microtubule-associated protein 1 light chain 3B (LC3B), an autophagy marker, by ATF4 and acts as a survival mechanism. Hypoxia, UPR and autophagy markers (such as Pimonidazole, carbonic anhydrases IX (CAIX), C/EBP homologous protein (CHOP) and LC3B) were evaluated by immunohistochemical approach in spheroids, xenografts models and breast cancer samples. CHOP immunohistochemical staining was performed in breast cancer sections from a series of patients. CHOP was expressed in cells surrounding necrotic areas. No correlation were found with clinical outcome and further studies are needed.

Autophagy is a catabolic process of self-digestion that occurs at basal levels in all cells, degrading old and damaged components such as proteins and organelles. Autophagy is upregulated in response to cellular stress, including the types imposed on cancer cells, and its role in these circumstances is often to facilitate cell survival. However, the molecular mechanisms regulating autophagy in response to cellular stress are still not well understood. Other components of the cell that are regulated in response to cellular stress are small non-coding portions of RNA termed microRNAs (miRNAs) that act to control protein levels in the cell by degrading mRNA targets or repressing their translation. The overall objective of this research was to identify candidate miRNA regulators of autophagy. My hypothesis was that miRNAs that regulate autophagy will demonstrate differential expression in response to cell stresses that induce autophagy. To test this hypothesis, I conducted miRNA expression profiling by Illumina sequencing in untreated BT-474 breast cancer cells and in BT-474 cells treated with a high dose of tamoxifen, a known inducer of autophagy. 113 distinct miRNAs were found to be significantly differentially expressed (p<0.05 and >1.5 fold) between the tamoxifen treated sample and control, and the differential expression of a subset of these miRNAs was validated using QRT-PCR. Using the online miRNA resource, TargetScan, 27 of the differentially expressed miRNAs were found to have potential autophagy-related targets, and of these, seven were selected to undergo further expression analysis in two additional breast cancer cell-lines using an additional autophagy inducing treatment, nutrient deprivation. Four of the selected miRNAs demonstrated similar patterns of differential expression in all three breast cancer cell-lines under the two different autophagy-inducing conditions. These four miRNAs consistently showed decreased expression when autophagy was induced, and have the potential to be direct regulators of the autophagy pathway upon cell stress induction in breast cancer cells. Understanding the mechanisms underlying the regulation of stress-induced autophagy may provide insight into the role autophagy plays in breast cancer and reveal potential targets to alter the process for clinical benefit.

When breast tumor cells encounter stress due to cancer therapies, they may enter a dormant state, escaping from treatment-induced apoptosis. Dormant cells may eventually regain proliferative capabilities and cause recurrent metastatic disease, which is the leading cause of mortality in breast cancer patients. We sought to determine if a high dose of radiation therapy (RT) or combined chemo-immunotherapy, with and without the blockade of autophagy by chloroquine (CQ), could overcome treatment-induced tumor dormancy or relapse. We found that autophagy contributes in part to treatment-induced tumor dormancy. We also found that three therapeutic strategies were successful in inhibiting or preventing tumor relapse. These include: 18Gy/day RT, chemotherapy combined with the blockade of autophagy, and combined chemo-immunotherapy. Follow-up studies are needed to determine the feasibility of preventing tumor relapse by prolonging tumor dormancy versus eliminating dormant tumor cells.

Philosophiae Doctor - PhD
Breast cancer is one of the most common cancers among women in South Africa and the second leading cause of cancer death after lung cancer. According to the American Cancer Society 2015, women have a 12% chance of developing invasive breast cancer and a 3% chance of dying from it. Despite the wide variety of breast cancers e.g. lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS), many share the same etiology and target tissue. Estrogen related carcinogenesis with regard to breast cancer typically results from the activation of distinct signalling pathways. These pathways are not mutually exclusive and are often constituted by receptor mediated stimulation of cell proliferation caused by specific transcriptional gene activation, reactive oxygen species (ROS) formation causing DNA damage and consequently mutations. The molecular pathways that cause drug resistance are not fully understood and the search continues to find novel targets for treatment. The effects of non-toxic triterpenes, oleanolic acid and ursolic acid and the role of autophagy and apoptosis as mechanisms to overcome drug resistance in breast cancer were studied in vitro in MCF-7 breast cancer cells and MCF10A breast cells. In this study the first aim was to establish the influence of OA and UA on cell growth and to see if opposing proliferation patterns could observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells. This was followed by morphology studies to establish the possible presence of cytotoxicity and examination of molecular pathways contributing to the anti-cancerous properties of UA and OA and their validity as therapeutic agents. The MCF-7 breast cancer cell line and the immortalized normal mammary cell line, MCF-10A were treated with different concentrations of UA and OA for 6hrs, 12hrs, 24hrs, 48hrs, and 72hrs respectively. Cell morphology was studied in hematoxylin and eosin as well as Hoechst and acridine orange stained cells and viability was measured using crystal violet staining. Molecular techniques employed included the Tali® Apoptosis - and the cellROX assays, flow cytometry and western blotting. Morphological, viability and apoptotic studies have shown that at their lowest concentration, both UA and OA have anti-proliferative and apoptotic effects on MCF-7 and to a lesser extent on MCF-10A. Flow cytometric analysis of treated cells has demonstrated cell arrest in the S- and G2/M phase. The MCF-7 and MCF-10A cells growth inhibition effect may be due to increased autophagy and apoptosis as an alternative to decreased proliferation in MCF-7 cells. This possibility should be evaluated in further studies. The results showed that UA was more effective OA in decreasing cell numbers and it may be applied as treatment for breast cancer. Our observation has shown the treatment with OA and UA increased cell death in MCF-7 cells.The opposing proliferation patterns observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells could possibly be ascribed to ERß forming homodimers that may facilitate proliferation, whereas ERɑ/ß heterodimers (expressed in 59% of breast cancers) are frequently associated with the ERɑ antagonising actions of ERß. The results indicate a trend towards biphasic and anti- proliferative effects of the reactants in breast cancer cells which may contribute towards the development of anti- cancer therapies. However, further work is must be done to identify the OA and UA mechanism(s) responsible for anticancer activity.
Libyan Embassy

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Introduction: Among the cancers that afflict females world-wide, neoplastic disease of breast tissue is the most frequently diagnosed form and the leading cause of cancer-related death. Conventional treatment entails the use of doxorubicin, an anticancer agent belonging to the anthracycline family of chemotherapeutic drugs. Cancer cells are becoming increasingly resistant to doxorubicin therapy. The existence of hypoxic zones, which is a common feature of solid tumours, has been shown to promote the selection of therapy resistant clones in proliferating cancer cells. By modifying cellular homeostasis, neoplastic cells are capable of tolerating the hypoxic insult and thriving within the hostile microenvironment of the tumour. This adaptation is known as ‘the hypoxic response’ and is mediated through the action of the transcriptional regulator, HIF-1. Its expression in cancer tissue has been associated with a dismal prognosis as it promotes the degree of malignancy to an advanced stage. Hypothesis & Aims: We hypothesized that the targeting of HIF-1α would circumvent the ‘protective’ hypoxic response conferred upon breast cancer and improve the cytotoxicity of doxorubicin treatment. In this study, the first aim was to identify the hypoxic conditions at which the MCF-7 breast cancer cell line manifests a doxorubicin-resistant phenotype. This was followed by examination of the molecular pathways contributing to the hypoxic resistance by elucidating the potential relationship with the hypoxic regulator HIF-1α. Once the involvement of HIF-1α was established, the next aim was to evaluate whether the attenuation of HIF-1α would terminate the resistant phenotype and sensitize the neoplastic MCF-7 cells to doxorubicin treatment. Finally, the reproducibility of the in vitro experiment and efficacy of treatments within an animal model was evaluated. 2-Methoxyestradiol is a naturally occurring metabolite originating from 17β-estradiol. It has recently been exploited as an anticancer agent due to its anti-proliferative and anti-angiogenic properties. Among its various mechanisms of action, this compound has been shown to inhibit the expression of HIF-1α. It is for this reason that this study employed 2-methoxyestradiol in the adjuvant therapeutic treatment, along with doxorubicin. Methods: The in vitro experimental model employed the use of the breast adenocarcinoma estrogen receptor (ER-positive cell line, MCF-7. These neoplastic cells were propagated under standard culture conditions until reaching ~70-80% confluency, after which treatment commenced. The treatment regime comprised a 12 hour exposure to the doxorubicin (1 μM) chemotherapeutic agent, either alone or in combination with HIF-1α inhibitors, 2-methoxyestradiol (10 μM) or siRNA duplex (400 nM), with parallel incubations under normoxic (21%) and hypoxic (~0.1%) conditions. To serve as a positive control for HIF-1α expression, cells were treated with CoCl2 (100 μM). Molecular techniques employed included the Caspase-Glo® 3/7 Assay, western blotting, and the bioreductive MTT Assay. Mitochondrial integrity was assessed by live cell imaging/fluorescent microscopy. Cellular viability was monitored at all times. The experiment was then translated into a pre-clinical in vivo model where C57BL/6 mice bearing E0771 xenografts (4 week growth) were allocated into the following treatment groups: (1) control (2) doxorubicin (5 mg.kg-1), (3) 2-methoxyestradiol (45 mg.kg-1), and (4) the combination of the two previously mentioned groups. Body weight and the rate of tumour growth were monitored throughout the experiment. Results: Treatment with CoCl2 effectively stabilized HIF-1α under normoxic conditions. 2-Methoxyestradiol was capable of attenuating HIF-1α expression under both normoxia and hypoxia as compared with siRNA transfection, which was only effective under normoxia. HIF-1α stabilization was accompanied by an increase in autophagy along with the morphological transformation of mitochondria from an elongated network to shorter disc-like forms. On the other hand, HIF-1α attenuation caused an induction in the expression of the apoptotic markers, cleaved caspase 3 and cleaved PARP, as well as the restoration of the normoxic morphology. The exposure of MCF-7 cells to 1 μM doxorubicin for 12 hours produced a differential effect in the bioreductive MTT assay between normoxic and hypoxic conditions (42.97 ± 3.095% vs. normoxic dox, p<0.01), while stimulating the apoptotic and autophagic pathways. Compared to the control, a significant expression of phospho-AMPK became evident at 21% O2, while the levels remained stable at ~0.1% O2 after doxorubicin exposure. Furthermore, chemotherapeutic treatment caused the morphology of the mitochondria to appear dot-like. Although the combination of the two drugs removed the differential effect witnessed in the MTT assay, there was no significant change when compared to doxorubicin. Levels of apoptotic cell death decreased under both oxygen conditions. While HIF-1α and autophagy decreased under normoxia, they remained elevated under hypoxia. In the in vivo component of the study, the administration of doxorubicin and 2-methoxyestradiol, alone or in combination, did not affect the rate of tumour growth or induce systematic toxicity in any of the experimental mice. When drugs were administered separately, a decrease in apoptosis along with a concomitant increase in autophagy and p-AMPK expression became noticeable while neither treatment had any significant effect on the expression of HIF-1α. Adjuvant administration, however, was capable of attenuating HIF-1α along with autophagy. Discussion: By inducing (CoCl2) and inhibiting (2-methoxyestradiol; siRNA duplex) HIF-1α, it was established that the autophagic pathway in the in vitro experimental setting of this study was dependent on the expression of HIF-1α. The bioreductive MTT assay measures the metabolic state of a cell, which is an indirect indication of cellular viability. Based on this, hypoxia was shown to confer survival to neoplastic MCF-7 cells based on the differential effect witnessed after doxorubicin treatment. Apart from the induction of apoptosis and its associated mitochondrial fragmentation, the chemotherapeutic drug increased the activation of the metabolic sensor, AMPK, which upregulated autophagy during normoxia. While this autophagic process may assist in the killing mechanism, we speculate that the autophagy upregulated under hypoxia may be responsible for the survival effect and is most likely dependent on HIF-1α. In contrast to eliciting a synergistic cytotoxic effect, the combination of doxorubicin with 2-methoxyestradiol produced an antagonistic effect on cellular viability instead. We propose that under normoxia, the combined treatment may stimulate the MCF-7 neoplastic cells to enter a state of growth arrest, or senescence, since the results indicate that the decrease in HIF-1α-dependent autophagy did not significantly affect cellular viability. Under hypoxia, despite the incorporation of the pharmacological HIF-1α inhibitor (2-methoxyestradiol), the expression levels of HIF-1α remained unaffected. We speculate that this could be the result of a potentiated stabilization of HIF-1α caused by the build-up of ROS and TCA intermediates which may be the outcome of mitochondrial dysfunction inflicted upon adjuvant therapy under hypoxia. Furthermore, it is also likely that the slight mitogenic effect observed within the MTT assay may be caused by the conversion of 2-methoxyestradiol to a chemically-reactive estrogen derivative, possibly by the action of doxorubicin, and the fact that an ER-positive cancer cell line was employed in this study. With regards to the in vivo experimental model, we speculated that the failure of the molecular changes to manipulate the growth of the tumour could have been the result of an ineffective time- and/or dose regime. Conclusion: We therefore reject our hypothesis based on the fact that an antagonistic rather than synergistic effect was witnessed when the tumorigenic MCF-7 cell line was treated with adjuvant therapy. The results warrant the need for extensive testing on the pharmacodynamics of 2-methoxyestradiol, and more informative techniques to compliment the study.
AFRIKAANSE OPSOMMING: Inleiding: Borskanker is die mees algemeen gediagnoseerde kanker asook die hoof oorsaak van kanker-verwante sterftes in vrouens wêreldwyd. Konvensionele behandeling behels die toediening van doxorubicin, ‘n anti-kankermiddel wat aan die antrasiklien-familie van chemoterapeutiese middels behoort. Kankerselle begin egter toenemend weerstandbiedend raak teen doxorubicin behandeling. Daar is al bewys dat die voorkoms van hipoksiese sones, wat ‘n algemene eienskap van soliede tumore is, die seleksie vir weerstandbiedende klone van prolifererende kankerselle, veroorsaak. Neoplastiese selle kan hierdie hipoksiese toestande weerstaan en in hierdie ongunstige mikro-omgewing floreer deur sellulêre homeostase te modifiseer. Hierdie aanpassing staan bekend as die ‘hipoksiese respons’ en word bemiddel deur die aksies van die transkripsiefaktor reguleerder, HIF-1. Die verhoogde uitdrukking van HIF-1 in kankerweefsel word oor die algemeen geassosieer met ‘n swak prognose omdat dit die maligniteit vehoog. Hipotese en Doelwitte: Die hipotese van hierdie studie behels dus die volgende: Deur HIF-1α te inhibeer, sal die ‘beskermende’ hipoksiese respons wat in borskankerselle voorkom omseil kan word en sodoende die sitotoksisiteit van doxorubicin terapie verhoog. Die eerste doelwit van hierdie studie was dus om die hipoksiese kondisies te identifiseer waar MCF-7 selle ‘n doxorubicin-weerstandbiedende fenotipe vertoon. Daarna is die molekulêre paaie wat bydrae tot hierdie hipoksiese weerstand ondersoek asook hul moontlike verwantskap met die hipoksiese reguleerder, HIF-1α. Nadat die rol van HIF-1α bevestig is, was die volgende doelwit om te bepaal of die inhibisie van HIF-1α die weerstandbiedende fenotipe sal onderdruk en neoplastiese MCF-7 selle sal sensitiseer vir doxorubicin behandeling. Laastens is die herhaalbaarheid en effektiwiteit van behandeling in die in vitro eksperimente ook in ‘n diermodel getoets. 2-Methoxyestradiol is ‘n metaboliet van 17β-estradiol wat natuurlik in die liggaam voorkom. Dit is ook onlangs as ‘n anti-kanker middel geïdentifiseer as gevolg van die anti-verdelende en anti-angiogeniese eienskappe. Een van die eienskappe van 2-methoxyestradiol is dat dit ook die uitdrukking van HIF-1α kan onderdruk. Dit is dan ook vir hierdie rede dat 2-methoxyestradiol in hierdie studie as bykomende terapie saam met doxorubicin gebruik is. Metodes: Die in vitro eksperimentele model behels die gebruik van ‘n borsadenokarsinoom, estrogeenreseptor (ER)- positiewe sellyn, MCF-7. Hierdie neoplastiese selle is onder standaard weefselkultuur omstandighede gekweek totdat konfluensie van ~70-80% bereik is, waarna behandeling begin het. Die behandelingsprosedure behels ‘n 12 uur blootstelling aan doxorubicin (1 µM) chemoterapeutiese middel alleen of in kombinasie met die HIF-1α inhibitore, 2-methoxyestradiol (10 µM) of siRNA duplex (400 nM) in normoksiese (21% O2) en hipoksiese (~0.1% O2) toestande. Die selle is ook met CoCl2 behandel wat gedien het as ‘n positiewe kontrole vir HIF-1α uitdrukking. Molekulêre tegnieke wat tydens hierdie studie gebruik is, sluit die “Caspase-Glo® 3/7” bepaling in, asook die westelike kladtegniek en die MTT bepaling. Mitochondriale integriteit is bepaal deur middel van lewende sel afbeeldings/fluoresensie mikroskopie. Sellewensvatbaarheid is ten alle tye gemonitor. Hierdie eksperment is verder ook in ‘n pre-kliniese in vivo model uitgevoer waar C57BL/6 muise met E0771 xenografte (4 weke groei) geïnduseer is en in die volgende behandelingsgroepe verdeel is: (1) kontrole; (2) doxorubicin (5 mg.kg-1); (3) 2-methoxyestradiol (45 mg.kg-1); en (4) die kombinasie van laasgenoemde twee groepe. Die liggaamsgewig en die tempo van tumorgroei is tydens die hele eksperiment gemonitor. Resultate: CoCl2 behandeling het HIF-1α effektief gestabiliseer tydens normoksiese omstandighede. 2-Methoxyestradiol het HIF-1α uitdrukking tydens normoksiese en hipoksiese toestande onderdruk wanneer dit vergelyk is met siRNA transfeksie wat slegs tydens normoksiese toestande effektief was. HIF-1α stabilisering het gepaardgegaan met ‘n toename in autofagie asook morfologiese veranderinge in die mitochondria vanaf ‘n verlengde netwerk tot korter skyfagtige vorme. Aan die ander kant het HIF-1α onderdrukking ‘n toename in die apoptotiese merkers, nl kliewing in caspase-3 and PARP veroorsaak wat gepaard gegaan het met die herstel van die tubulêre mitochondriale netwerk. Die blootstelling van die MCF-7 selle aan 1 µM doxorubicin vir 12 ure het ‘n differensiële effek in die bioreduktiewe MTT bepaling tot gevolg gehad tussen normoksiese en hipoksiese toestande (42.97 ± 3.095%, p<0.1), terwyl die apoptotiese- en autofagiese paaie in beide toestande gestimuleer is. ‘n Insiggewende toename in fosfo-AMPK uitdrukking was sigbaar tydens normoksiese toestande van 21% O2, terwyl dit onveranderd gebly het tydens hipoksiese toestande van 0.1% ~O2 na doxorubicin behandeling. Die morfologie van die mitochondria het ‘n ‘kollerige’ voorkoms tydens doxorubicin behandeling gehad. Alhoewel die behandeling van die selle met beide middels gelyktydig, die differensiële effek soos weerspieël in die MTT bepaling ophef, is daar geen insiggewende verandering wanneer met doxorubicin behandeling vergelyk word nie. Apoptotiese seldood verminder met gelyktydige behandeling van biede middels tydens normoksiese en hipoksiese toestande. HIF1-α en autofagie het afgeneem tydens normoksiese toestande, maar bly vehoog tydens hipoksie. In die in vivo model, het die toediening van doxorubicin en 2-methoxyestradiol alleen en in kombinasie nie tumorgroei geaffekteer nie en ook nie sistemiese toksisiteit in enige van die eksperimentele muise tot gevolg gehad nie. Die afsonderlike toediening van die middels het ‘n afname in apoptose in ‘n toename in autofagie en p-AMPK uitdrukking tot gevolg gehad, terwyl afsonderlike toediening van die middels nie ‘n effek op HIF-1α uitdrukking gehad het nie. Die gelyktydige toediening van biede middels het egter ‘n onderdrukking van HIF1-α teweeggebring. Bespreking: Deur HIF-1α te induseer (CoCl2) en te inhibeer (2-methoxyestradiol en siRNA) in hierdie in vitro eksperimentele omstandighede, bevestig hierdie resultate dat autofagie afhanklik is van die uitdrukking van HIF-1α. Die bioreduktiewe MTT bepaling meet die metaboliese staat van die sel wat indirek sellewensvatbaarheid bepaal. Gebasseer op hierdie bepaling is bewys dat hipoksie ‘n weerstandbiedende fenotipe veroorsaak teen doxorubicin behandeling in neoplastiese MCF-7 selle. Doxorubicin veroorsaak ‘n toename in apoptose met geassosieerde mitochondriale fragmentering asook ‘n aktivering van die metaboliese sensor, AMPK, wat autofagie stimuleer in normoksiese omstandighede. Alhoewel ‘n toename in autofagie seldood kan stimuleer, spekuleer ons dat ‘n toename in autofagie tydens hipoksie verantwoordelik kan wees vir seloorlewing wat heel moontlik ook afhanklik van HIF-1α is. In kontras met die verwagting dat die kombinasie behandeling ‘n sinergistiese sitotoksiese effek sou teweegbring, dui ons resultate dat daar ‘n antagonistiese effek op sellewensvatbaarheid was. Ons stel voor dat die gekombineerde behandeling tydens normoksiese toestande MCF-7 neoplastiese selle stimuleer om in ‘n toestand van groeistaking in te gaan aangesien die resultate daarop dui dat ‘n afname in HIF-1α afhanklike autofagie nie sellulêre lewensvatbaarheid beïnvloed het nie. Tydens hipoksie, ten spyte van die bykomdende behandeling met die HIF-1α inhibitor (2-methoxyestradiol), het die vlakke van HIF-1α onveranderd gebly. Ons spekuleer dat dat dit die gevolg kan wees van die stabilisering van HIF-1α as gevolg van ‘n toename in ROS en TCA intermediate wat die gevolg van mitochondriale wanfunksie kan wees tydens bykomende terapie onder hipoksiese toestande. Dit is ook moontlik dat die mitogeniese effek wat waargeneem is met die MTT bepaling die gevolg kan wees van die omsetting van 2-methoxyestradiol na ‘n chemiese-reaktiewe estrogeen derivaat; moontlik as gevolg van die aksie van doxorubicin en die feit dat die sellyn wat in hierdie studie gebruik is, ‘n ER-positiewe kankersellyn is. Met verwysing na die in vivo eksperimentele model, spekuleer ons dat die molekulêre veranderinge wat nie in die tumorgroei weerspieël word nie, die resultaat van oneffektiewe tyds- en dosis behandelingswyses is, of foutiewe toediening van die middel kan wees. Gevolgtrekking: Ons verwerp dus ons hipotese gebaseer op die feit dat bykomende (adjuvante) behandeling eerder ‘n antogonistiese effek as ‘n sinergistiese effek op seldood in MCF-7 selle het. Hierdie resultate regverdig die nodigheid van intensiewe toetsing op die farmakodinamika van 2-methoxyestradiol asook die gebruik van meer informatiewe tegnieke om hierdie studie te komplimenteer.
CANSA and Marie Stander

Despite clinical and pharmacological advancement in medical science breast cancer has become a global concern due to the high mortality rate. Breast cancer is mainly associated with altered redox status, cell cycle, chronic inflammation, and increased proliferative rate. Breast cancer has various molecular subtypes and adequate knowledge of these altered cell cycle regulatory cascades and molecular subtypes of breast cancer is a must for proper prognosis and its successful treatment. The discovery of drugs with anticancer properties, particularly against the specific subtype of breast cancer has become a challenging task for cancer researchers. Dietary polyphenolic compounds as cancer chemopreventive agents have drawn much attention among researchers because polyphenolic compounds are natural in origin with lesser side effects and have a wide range of action against various subtypes of breast cancer. Dietary compounds with antioxidant properties have been reported to act on an array of genes and proteins associated with breast cancer pathogenesis and thus regulate the signaling cascade related to autophagy, chronic inflammation, apoptosis, and cell cycle regulation. All in all, these natural compounds regulate growth and progression of a tumour with less or no side effects. Thus, the current article focuses primarily here on various aspects of breast cancer and food polyphenolic compounds as wellas their molecular mechanism for managing breast cancer.