Hepatocellular carcinoma (HCC) is the most common liver malignancy that usually develops in patients suffering from chronic liver diseases. One of the major problems faced in the treatment of HCC is severe chemoresistance. Endoplasmic reticulum (ER) stress and hepatic stellate cells play an important role in tumour survival and growth as well as fibrosis. This study further investigates the crosstalk between ER-stress and hepatic stellate cells in HCC resistant cells as well as their relation to chemoresistance markers expression. Mice with chemically induced HCC were divided in 3 different treatment group; one was only treated with doxorubicin, one only with pharmacological ER-stress inhibitor 4μ8C, and one was treated with a combination of doxorubicin and 4μ8C. Tumour burden, fibrosis and cell proliferation were assessed through histological analysis and ImageJ processing. Chemoresistance markers expression was evaluated through mRNAs determination using real-time qPCR. While the combined treatment consisting of doxorubicin and pharmacological ER-stress inhibitor (4μ8C) has shown to positively reduce tumour progression, ferroptosis and collagen deposition, consequently decreasing fibrosis, drug resistance markers’ expression, on the other hand, seems to be more intricate, thus indicating that further investigations are probably needed. / Hepatocellulärt karcinom (HCC) är den vanligaste maligniteten i levern som vanligtvis utvecklas hos patienter som lider av kroniska leversjukdomar. Ett av de största problemen vid behandling av HCC är svår kemoresistens. Stress i endoplasmatiska retikulum (ER) och hepatiska stellatceller spelar en viktig roll för tumörernas överlevnad och tillväxt samt för fibros. I denna studie undersöks vidare samspelet mellan ER-stress och hepatiska stellatceller i HCC-resistenta celler samt deras relation till uttryck av kemoresistensmarkörer. Möss med kemiskt inducerad HCC delades in i tre olika behandlingsgrupper; en behandlades enbart med doxorubicin, en enbart med den farmakologiska ER-stresshämmaren 4μ8C och en behandlades med en kombination av doxorubicin och 4μ8C. Tumörbörda, fibros och cellproliferation bedömdes genom histologisk analys och ImageJ-bearbetning. Kemoresistensmarkörernas uttryck utvärderades genom bestämning av mRNA med hjälp av qPCR i realtid. Medan kombinationsbehandlingen bestående av doxorubicin och farmakologisk ER-stresshämmare (4μ8C) har visat sig minska tumörprogressionen, ferroptos och kollagenavlagring och därmed minska fibros, verkar uttrycket av läkemedelsresistensmarkörer å andra sidan vara mer invecklat, vilket tyder på att det troligen behövs ytterligare undersökningar.

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

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction The discovery of Doxorubicin in the 1960s has drastically improved the survival rates of cancer patients, however, its success is limited by dose-dependent cardiotoxicity. While much of the literature has focused on acute cardiotoxicity which is minor and generally reversible, chronic cardiotoxicity poses a serious threat to cancer survivors since it can lead to dilative cardiomyopathy, congestive heart failure and even death. The mechanisms that contribute to cardiotoxicity are still a matter of controversy, however, oxidative stress-induced myocardial damage and apoptosis are thought to be the major role players. Reperfusion injury, also characterized by oxidative stress and apoptosis, occurs as a result of restoring blood flow to an ischemic heart. Fortunately, pre- and post-conditioning are techniques employed to minimize this damage and are thought to do so by activating the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways. The RISK pathway involves the pro-survival kinases, Erk1/2 and Akt, while the SAFE pathway, triggered by TNF-α, involves Jak2 and STAT3. Since both reperfusion injury and Doxorubicin-induced cardiotoxicity share similar characteristics, this study aimed to determine whether the RISK and SAFE pathways are activated in response to long-term Doxorubicin treatment. Furthermore, this study aimed to determine whether TNF-α is produced during treatment, since its role in Doxorubicin-induced cardiotoxicity is still relatively unknown. Methods H9c2 cardiomyocytes and differentiated C2C12 myotubes were treated daily with increasing concentrations of Doxorubicin for a total of 120 hours. Cell viability, apoptosis and necrosis were assessed using the MTT, Caspase-Glo® 3/7 and lactate dehydrogenase assays respectively. TNF-α production was measured using Quantikine® ELISA kits and various assays were used to assess oxidative stress, anti-oxidant capacity and anti-oxidant status. The protein expression of the RISK and SAFE pathways were analysed by western blotting using both phospho-specific and total antibodies. Results and Discussion Treatment with Doxorubicin caused a time- and dose-dependent decrease in cell viability in both cell lines and this was accompanied by an increase in apoptosis. In the H9c2 cardiomyocytes, treatment with 0.2 μM Doxorubicin yielded significant levels of TNF-α after 120 hours and we can speculate that these low levels partially protected the cells from the toxic effects of Doxorubicin by activating the SAFE pathway, since both Jak2 and STAT3 were phosphorylated at this concentration. Treatment with 1 μM Doxorubicin caused a larger and biphasic pattern of TNF-α release, which may have then contributed to the decrease in cell viability, since the SAFE pathway was not activated at this concentration. Akt was phosphorylated during the first 72 hours of treatment with the low dose of Doxorubicin, but chronic treatment prevented this phosphorylation. While Erk1/2 was not phosphorylated at all at the low dose of Doxorubicin, neither Akt nor Erk1/2 was phosphorylated at the high dose and their inhibition may contribute to the cardiotoxic effects of Doxorubicin. In the C2C12 myotubes, a significant amount of TNF-α was produced after 120 hours of treatment with the low dose of Doxorubicin. Treatment with the high dose of Doxorubicin induced significant TNF-α production at every time point. While STAT3 was phosphorylated at the serine residue after treatment with the low dose of Doxorubicin, treatment with the high dose induced phosphorylation at the tyrosine residue in a time-dependent manner. p-Jak2 expression was significantly down-regulated at both concentrations of Doxorubicin, suggesting that STAT3 proteins can by-pass activation by Jak2. The Erk1/2 leg of the RISK pathway was also not activated for the majority of the treatment period, however, p-Akt expression was increased at the low concentration of Doxorubicin relative to total Akt expression. Conclusion These observations indicate that treatment with Doxorubicin causes a severe, dose-dependent loss in viability which is likely to mediated by high concentrations of TNF-α (induced by high concentrations of Doxorubicin) and down-regulation of protective signaling pathways. TNF-α may confer partial protection at low concentrations by activating the SAFE pathway. However, activation of the SAFE pathway could not provide sufficient protection from Doxorubicin, most probably because the RISK pathway was not simultaneously activated. Our results also clearly highlight the differences between acute and chronic treatment since a single high dose of Doxorubicin produced vastly different responses to cumulative treatment with a low dose. Before one can extrapolate these results into the clinical setting, further research is required to provide a better understanding of the RISK and SAFE pathways and whether stimulation thereof will provide a protective effect. In addition, although our study has shown that TNF-α is produced in response to Doxorubicin treatment, its true role, whether beneficial or detrimental, remains to be determined. / AFRIKAANSE OPSOMMING: Inleiding Die ontdekking van Doksorubisien (DOKS) in die 1960’s het die oorlewingsyfer van kankerpasiënte drasties verhoog, maar DOKS-gebruik gaan egter ook gepaard met dosis-afhanklike kardiotoksisiteit. Terwyl die literatuur grootliks fokus op akute kardiotoksisiteit, wat minimaal en algemeen omkeerbaar is, hou kroniese kardiotoksisiteit ‘n ernistige bedreiging vir kankeroorlewendes in, aangesien dit kan lei tot dilatiewe kardiomiopatie, kongestiewe hartversaking, en selfs dood. Die spesfikieke meganismes wat bydrae tot kardiotoksisiteit is tans steeds onbekend, maar oksidatiewe stres-geinduseerde miokardiale skade en apoptose word beskou as hoof bydraende faktore. Reperfussie skade, ook gekaraktiseer deur die teenwoordigheid van oksidatiewe stres en apoptose, kom voor as gevolg van die herstel van bloedtoevoer na ‘n isgemiese hart. Om die skade te minimaliseer word voor- en nakondisionerings tegnieke geïmplimenteer wat die RSHK (Reperfussie Skade Herwinnings Kinase) en OAFV (Oorlewerings Aktiverings Faktor Versterkings)-weë aktiveer. Die RSHK weg maak gebruik van pro-oorlewings kinases Erk1/2 en Akt, terwyl die TNF-α geaktiveerde OAFV weg Jak2 en STAT3 betrek. Aangesien beide reperfussie skade en DOKS-geinduseerde kardiotoksisiteit soortgelyke eienskappe deel, is die doel van hierdie studie om vas te stel of die RSHK en OAFV-weë geaktiveer word in langtermyn DOKS behandeling. Boonop is nog ‘n doel van hierdie studie om vas te stel of TNF-α geproduseer word tydens behandeling, aangesien die rol daarvan in DOKS-geinduseerde kardiotoksisiteit steeds onbekend is. Metodes H9c2 kardiomiosiet en gedifferensieerde C2C12 miobuise was daagliks behandel met toenemende konsentrasies van Dox vir 120 ure. Die effekte van DOKS op sel lewensvatbaarheid, apoptose en nekrose is onderskeidelik ondersoek deur middel van die MTT, Caspase-Glo® 3/7 en LDH toetse. TNF-α produksie is bepaal deur van die Quantikine® toets gebruik te maak, en verskeie metodes is gebuik om die oksidatiewe stres, anti-oksidantkapasiteit en anti-oksidantstatus te bepaal. Die proteïenuitdrukking van die RSHK (Erk1/2 en Akt) en OAFV (Jak2 en STAT3) weë was ontleed deur middel van westerse afklattingstegniek deur van beide fosfospesifieke en totale teenliggaampies gebruik te maak. Resultate en Bespreking Behandeling met DOKS het ‘n tyd en dosis-afhanklike afname in sel lewensvatbaarheid in beide sellyne veroorsaak, wat gepaard gegaan het met ‘n toename in apoptose. In die H9c2 kardiomiosiete, het ‘n lae DOKS dosisbehandeling (0.2 μM) betekenisvolle vlakke van TNF-α na 120 uur opgelewer en ons kan spekuleer dat hierdie lae vlakke gedeeltelik die selle van die toksiese effekte van DOKS deur die aktivering van die OAFV weg beskerm het omrede beide Jak2 en STAT3 by hierdie konsentrasie gefosforileer is. Die hoë DOKS dosis (1 μM) het ‘n groter en bifasiese patroon van TNF-α vrystelling vertoon, wat kon bydra tot die DOKS-geinduseerde afname in sel lewensvatbaarheid. Akt is gedurende die eerste 72 uur van behandeling gefosforileer met die lae DOKS dosis, maar kroniese behandeling het hierdie fosforilering verhoed. Terwyl Erk1/2 glad nie gefosforileer is by die lae DOKS dosis nie, is nie Akt of Erk1/2 by die hoë dosis gefosforileer nie, en kan hierdie inhibering bydrae tot die kardiotoksiese effekte van DOKS. In die C2C12 miobuise, is ‘n betekenisvolle hoeveelheid TNF-α na 120 uur van behandeling geproduseer by die lae DOKS dosis. Behandeling met die hoë DOKS dosis het betekenisvolle TNF-α produksie geinduseer by elke tydspunt. Terwyl STAT3 gefosforileer is by die serienresidu na behandeling met die lae DOKS dosis, het behandeling met die hoë dosis fosforilering by die tirosienresidu op ’n tydsafhanklike wyse plaasgevind. p-Jak2 uitdrukking was betekenisvol verminder by beide DOKS konsentrasies, wat aanduidend is dat die STAT3 proteïene nie geaktiveer hoef te word deur Jak2 nie. Die Erk1/2 been van die RSHK weg is ook nie geaktiveer gedurende die oorhoofse behandelingstydperk nie, alhoewel, p-Akt wel uitgedruk is by die lae konsentrasie van DOKS relatief tot die totale Akt uitdrukking. Gevolgtrekkings Die resultate van hierdie studie toon dat DOKS-behandeling tot ‘n dosis-afhanklike verlies in sel lewensvatbaarheid lei. Hierdie effek word waarskynlik bemiddel deur die teenwoordigheid van hoë konsentrasies TNF-α, en ook die afregulering van die beskermende seinweë. TNF-α kan moontlik gedeeltelike beskerming bied by lae konsentrasies deur aktivering van die OAFV weg. Die aktivering van die OAFV weg kon egter nie voldoende beskerming teen DOKS bied nie; moontlik as gevolg van die afwesigheid van die gelyktydige RSHK weg aktivering. Ons resultate vertoon die verskille tussen die akute en kronies behandeling aangesien ‘n enkele hoë-dosis van DOKS, in vergelyking met ‘n kumulatiewe lae-dosis, grootliks verskillende resultate opgelewer het. Voordat hierdie resultate klinies verder ondersoek kan word is verdere navorsing nodig om TNF-α en die RSHK en OAFV-weë beter te verstaan, en om vas te stel of stimulering van hierdie seinoordragpaaie ‘n beskermende effek teweeg sal bring.

Η αντικαρκινική ένωση δοξορουβικίνη χρησιμοποιείται ευρέως είτε μόνη της είτε σε συνδυασμό με άλλα αντικαρκινικά φάρμακα, στην αντιμετώπιση του καρκίνου του μαστού, των ωοθηκών, του πνεύμονα αλλά και σε περιπτώσεις οξείας λευχαιμίας και σαρκωμάτων. Σε προηγούμενη έρευνα που πραγματοποιήθηκε στο εργαστήριο μας, σε λεμφοκύτταρα ανθρώπου αλλά και στην κυτταρική σειρά ποντικού C2C12, βρέθηκε ότι επάγει το σχηματισμό μικροπυρήνων ως αποτέλεσμα κυρίως χρωμοσωματικής θραύσης. Στην παρούσα εργασία, διερευνήθηκε περαιτέρω η ικανότητα της δοξορουβικίνης να προκαλεί θραύση του γενετικού υλικού καθώς και η ικανότητά της να επάγει τη διαδικασία της απόπτωσης σε λευχαιμικά κύτταρα ανθρώπου HL-60. Η μελέτη του κερματισμού του DNA λόγω της δράσης της δοξορουβικίνης πραγματοποιήθηκε με τη μέθοδο ηλεκτροφόρησης μοναδιαίων κυττάρων σε πηκτή αγαρόζης (SCGE) κάτω από αλκαλικές συνθήκες. Εκτιμήθηκαν οι παράμετροι tail length, % DNA in tail, tail moment, και olive tail moment που αποκαλύπτουν θραύση του DNA. Επιπρόσθετα, η ικανότητα της δοξορουβικίνης να προκαλεί θραύση του γενετικού υλικού αναλύθηκε μέσω δημιουργίας κλάσεων με ελάχιστη έως μέγιστη βλάβη. O μηχανισμός με τον οποίο προκαλεί θραύση του DNA διερευνήθηκε με τη χρήση της μεθόδου ηλεκτροφόρησης μοναδιαίων κυττάρων σε πηκτή αγαρόζης κάτω από αλκαλικές συνθήκες σε συνδυασμό με τη χρήση των επιδιορθωτικών ενζύμων Fpg και hOOG1. Μετέπειτα, εξετάστηκε η ικανότητα της δοξορουβικίνης να επάγει την απόπτωση. Η μελέτη πραγματοποιήθηκε με τη μέθοδο αναστολής της κυτταροκίνησης (CBMN). Επίσης, με τη μέθοδο CBMN πραγματοποιήθηκε μελέτη του φαινομένου της χρωμοσωματικής θραύσης. Για την περαιτέρω διερεύνηση του μηχανισμού με τον οποίον η υπό εξέταση χημική ένωση επάγει τη διαδικασία της απόπτωσης, αναλύθηκε η ικανότητά της να τροποποιεί την έκφραση της κασπάσης-3, μιας πρωτεΐνης που παίζει σημαντικό ρόλο στον καταρράκτη των μοριακών γεγονότων που εμπλέκονται στην ενεργοποίηση του προγραμματισμένου κυτταρικού θανάτου. Για το σκοπό αυτό εφαρμόστηκε η μέθοδος ανοσοαποτύπωσης της παραπάνω πρωτεΐνης (Western blot). Με βάση τα ευρήματά μας, η δοξορουβικίνη παρουσιάζει θραυσματογόνο δράση, όπως φάνηκε από την αύξηση της εξόδου του DNA από τους πυρήνες των κυττάρων μετά από ηλεκτροφόρηση σε αλκαλικές συνθήκες. Η δημιουργία των ρηγμάτων είναι ισχυρότερη σε χαμηλές συγκεντρώσεις. Η ικανότητα της δοξορουβικίνης να προκαλεί θραύση του γενετικού υλικού σχετίζεται με την οξείδωση των βάσεων του DNA, η οποία έχει ως αποτέλεσμα τη δημιουργία ασταθών σε αλκαλικές συνθήκες θέσεων (alkali labile sites). Επίσης, προκαλεί οριακή αύξηση της συχνότητας των μικροπυρήνων στις χαμηλότερες συγκεντρώσεις που μελετήθηκαν, ενώ δε φαίνεται να επάγει τη δημιουργία των μικροπυρήνων στις υψηλότερες συγκεντρώσεις. Το εύρημα αυτό είναι σε συμφωνία με την ιδιότητά της να προκαλεί μεγαλύτερη συχνότητα ρηγμάτων του DNA στις μικρές συγκεντρώσεις. Επιπρόσθετα, επάγει τη διαδικασία της απόπτωσης. Η επαγωγή αυτή είναι ισχυρότερη σε υψηλές συγκεντρώσεις και δικαιολογεί τις μειωμένες συχνότητες ρηγμάτων και μικροπυρήνων στις συγκεντρώσεις αυτές. Η κασπάση 3 συμμετέχει στην επαγόμενη από τη δοξορουβικίνη απόπτωση όπως φάνηκε από την αύξηση της έκφρασης της κασπάσης 3, μετά από ανάλυση κατά western, σε κυτταρικές καλλιέργειες που αναπτύχθηκαν παρουσία δοξορουβικίνης. / The anticancer drug doxorubicin is widely used, either alone or in combination with other anticancer drugs, in the treatment of solid tumours of the breast, lung, ovary, as well as in acute leukemia and sarcomas. Previous research in our laboratory showed that doxorubicin is able to induce micronucleus generation, in human lymphocytes and mouse cell line C2C12, mainly due to chromosome breakage. In the present study we investigated the clastogenic activity of doxorubicin as well as its ability to induce apoptosis. Ηuman leukemic cells HL-60 were chosen as the cell system to proceed our research. The clastogenic activity of doxorubicin was investigated by alkaline Single Cell Gel Electrophoresis (SCGE assay-Comet assay). Four parameters were analyzed, tail length, % DNA in tail, tail moment, and olive tail moment, which reveal DNA breakage. Additionally the capacity of doxorubicin to induce DNA fragmentation was analyzed by stratifying the cells into five classes with various degrees of DNA damage, from undamaged DNA to severely damaged DNA. The mechanism by which doxorubicin exerts its clastogenic activity was studied by enzyme linked comet assay. We used Fpg and hOOG1 DNA glycosylases. The ability of doxorubicin to induce apoptosis was studied by Cytokinesis-block Micronucleus assay. Also, the CBMN assay was used to assess the micronucleation on HL-60 due to the action of doxorubicin. To further elucidate the mechanism by which doxorubicin induce apoptosis we examined the ability of this compound to alter the expression of caspase 3, that plays a key role in the cascade of molecular events of programmed cell death. This analysis was performed by Western blot. Our findings indicate that doxorubicin exerts clastogenic activity as it provokes DNA migration from the nucleus after SCG electrophoresis in alkaline conditions. The generation of breaks on DNA strands seems to be more potent at low concentrations. The ability of doxorubicin to induce fragmentation of genetic material is correlated with the oxidation of DNA bases resulting in the formation of alkali labile sites. Furthermore, it induces marginal increase in the frequency of micronuclei at lower concentrations, while it doesn’t seem to induce micronucleation at higher concentrations. This finding is in accordance with the ability of doxorubicin to generate higher frequency of DNA breaks at low concentrations. Additionally, doxorubicin induces apoptosis. This induction is more potent at higher concentrations and is consistent with the reduced frequency of breaks and micronucleus generation at these concentrations. Activation of apoptosis due to doxorubicin treatment seems to be mediated by caspase 3.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressant effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the Glucocorticoid Receptor (GR), a cytosolic receptor that translocates to the nuclei upon ligand binding and alters gene transcription. Among a long list of genes activated by GCs is the Glucocorticoid Induced Leucine Zipper (GILZ). Although GC induced GILZ expression has been well established in lymphocytes, little is known whether cardiomyocytes respond to GCs by inducing GILZ. Unlike lymphocytes, in which GCs induce apoptosis and GILZ mediates GC induced apoptosis, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression pattern in cardiomyocytes in vivo and in vitro. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs and point to H9C2 cell line as a valid model for studying the biological function of GILZ in cardiomyocytes. I have also determined GILZ functions as GC induced cytoprotective protein against the known cardiac toxicant Doxorubicin. Finally I have determined GILZ stabilizes Bcl-xL pro-survival protein, providing a possible mechanism of cytoprotection in cardiomyocytes.

A cancer drug's effectiveness is contingent upon on its ability to reach all parts of the tumor. The distribution of drug in the tumor depends on several transport processes and depends on the physicochemical properties of the drug. These factors can lead to highly heterogeneous distributions of drug in the tumor interstitial space, leaving parts of the tumor unreached, and make it difficult to predict cellular exposure and understand its dependence on key system parameters. Theoretical models are powerful tools that can provide insight by simulating conditions that cannot be achieved or observed experimentally. Here, a Green's function method is utilized to simulate three-dimensional time-dependent diffusion and uptake of drugs in solid tumors with realistic vascular geometry. Regimes dependent on the time scales for transport are used to determine whether spatial and temporal effects must be resolved to predict cellular exposure. Simulations are performed to show the relationship between the plasma pharmacokinetics and cellular exposure for these regimes. Steep gradients in concentration arise when time scales for diffusion and uptake are comparable, implying that models based on well mixed compartments are inaccurate. Effects of linear and nonlinear kinetics of drug uptake on cellular exposure are demonstrated. The drug doxorubicin is commonly used against solid tumors. Cellular exposure to doxorubicin is complicated in vivo by its transport and physicochemical properties. The Green's function method is used to describe the in vivo transport and kinetics of doxorubicin, using parameters derived from in vitro results. Simulations show agreement with observed in vivo distributions of doxorubicin in tumor tissue as well as in vitro kinetics, and provide a link between the two types of experimental observations. The method is applied to the class of cancer drugs called antibody-drug conjugates (ADCs) which consist of a humanized antibody conjugated to extremely toxic small molecular weight drugs. ADCs exhibit complex in vivo kinetics dependent on many design parameters. A phenomenon exhibited by ADCs is the bystander effect, i.e. non-targeted cell killing, which is difficult to analyze based on in vivo observations. Simulations results agree with the observed in vivo distribution of ADCs in tumor tissue and with experimentally observed bystander effects. In summary, the the models presented here provide a novel approach for simulating the complex transport and cellular uptake kinetics exhibited by several cancer drugs. The models give a mechanistic basis for predicting cellular exposure to drugs which can aid, explain, and direct experimental approaches for improving cancer treatment.

INTRODUÇÃO: Cardiotoxicidade é frequentemente associada ao uso crônico de doxorubicina (DOX) podendo levar a cardiomiopatia e insuficiência cardíaca. A identificação de miRNAs cardiotoxicidade-específicos e seu potencial como biomarcadores poderia fornecer uma ferramenta prognostica valiosa e uma potencial área de intervenção. METODOLOGIA: Este é um sub-estudo do ensaio clínico prospectivo \"Efeito do Carvedilol na Prevenção da Cardiotoxicidade Induzida por Quimioterapia\" (ensaio CECCY) no qual incluiu 56 pacientes do sexo feminino (idade 49.9±3.3) provenientes do braço placebo. Os pacientes incluídos foram submetidos à quimioterapia com DOX seguido por taxanos. Troponina cardiaca I (cTnI), fração de ejeção do ventrículo esquerdo (FEVE) e microRNAs foram mensurados periodicamente. RESULTADOS: Os níveis circulantes de miR-1, -133b, -146a e -423-5p aumentaram significativamente durante o tratamento (18.6, 11.5, 10.6 e 12.1-vezes respectivamente; p < 0.001) enquanto miR-208a e -208b foram indetectáveis. cTnI aumentou de 6.6 ± 0.3 para 46.7 ± 5.5 pg/ml (p < 0.001) enquanto FEVE tendeu a diminuir de 65.3±0.5 para 63.8±0.9 (p=0.053) após 12 meses; deis pacientes (17.9%) desenvolveram cardiotoxicidade. miR-1 foi associado a mudanças na FEVE (r2=0.363, p < 0.001) enquanto miR-1 e -133b foram associados a cTnI (r2 = 0.675 e 0.758; p < 0.001). Além disso, miR-1 antecipou a cardiotoxicidade e mostrou uma area sobre a curva maior que cTnI para discriminar pacientes que desenvolveram cardiotoxicidade daqueles que não desenvolveram (AUC = 0.849 e 456, p<0.001 e 0.663, respectivamente). CONCLUSÃO: Nossos dados sugerem miR-1 como um potencial novo biomarcador de cardiotoxicidade induzida por DOX em pacientes com câncer de mama. Estes resultados podem levar a novas estratégias de detecção precoce do risco de lesão cardíaca induzida por DOX bem como a introdução de uma nova área para intervenção / INTRODUCTION: Cardiotoxicity is frequently associated with the chronic use of doxorubicin (DOX) and may lead to cardiomyopathy and heart failure. Identification of cardiotoxicity-specific miRNA biomarkers could provide clinicians with a valuable prognosis tool and a potential area for intervention. METHODS: This is an ancillary study from the prospective trial \"Carvedilol Effect in Preventing Chemotherapy-Induced Cardiotoxicity.\" (CECCY trial) which included 56 female patients (49.9±3.3 age) from placebo arm. Enrolled patients were treated with DOX followed by taxanes. Cardiac troponin I (cTnI), left ventricle ejection fraction (LVEF) and miRNAs were measured periodically. RESULTS: Circulating levels of miR-1, -133b, -146a and -423-5p increased along the treatment (18.6, 11.5, 10.6 and 12.1-fold respectively; p < 0.001); miR-208a and -208b were undetectable. cTnI increased from 6.6±0.3 to 46.7 ± 5.5 pg/ml (p < 0.001) while LVEF tended to decrease from 65.3±0.5 to 63.8±0.9 (p=0.053) over 12 months; ten patients (17.9%) developed cardiotoxicity. miR-1 was associated to changes in LVEF (r2=0.363, p < 0.001) while miR-1 and -133b were associated to cTnI (r2 = 0.675 and 0.758; p < 0.001). Furthermore, miR-1 anticipated cardiotoxicity and showed greater area under the curve than cTnI to discriminate between patients who did and did not developed cardiotoxicity (AUC = 0.849 and 456, p < 0.001 and 0.663, respectively). CONCLUSION: Our data suggest circulating miR-1 as a potential new biomarker of DOX-induced cardiotoxicity in breast cancer patients. These results may lead to new earlier strategies to detect drug-induced cardiac injury risk before it develops to an irreversible stage or introduce new area for intervention

O cubiuzeiro (Solanum sessiliflorum Dunal) é uma planta nativa da Amazônia, utilizada na medicina popular no tratamento de queimaduras e no controle da glicemia e colesterolemia. Análises fitoquímicas da polpa dos frutos do cubiuzeiro, conhecidos como maná-cubiu, revelaram que este apresenta em sua constituição carotenoides, compostos fenólicos e vitaminas. Devido aos poucos estudos sobre a atividade biológica do fruto e à presença de compostos com propriedades antioxidantes, os objetivos deste trabalho foram avaliar os efeitos da polpa do maná-cubiu sobre a estabilidade genômica, parâmetros de estresse oxidativo, expressão de genes pró-inflamatórios (iNos e COX-2) em ratos Wistar e determinar a presença de elementos químicos na polpa liofilizada por ICP-MS. Os animais receberam, por gavagem, a polpa liofilizada do maná-cubiu (125, 250, 375 ou 500 mg/kg p.c.) durante 14 dias. No último dia de tratamento foi administrada intraperitonealmente salina ou doxorrubicina (DXR, 16 mg/kg p.c.), usada como agente indutor de danos, e após 24 horas os animais foram eutanasiados. O sangue periférico e a medula óssea foram usados no teste do micronúcleo. No ensaio do cometa foram analisados fígado, coração e sangue periférico. Fígado e coração foram utilizados nas análises das substâncias reativas ao ácido tiobarbitúrico (TBARS) e glutationa reduzida (GSH) e na avaliação da expressão de RNAm dos genes COX-2 e iNos. Os resultados mostraram que a polpa do maná-cubiu não foi mutagênica nem citotóxica à medula óssea e ao sangue periférico dos animais. Nas doses 250 e 375 mg/kg p.c., a polpa do maná-cubiu reduziu o número de células micronucleadas induzidas pela DXR no sangue periférico, enquanto que na medula óssea somente a dose de 375 mg/kg p.c. foi antimutagênica. A polpa do maná-cubiu não foi genotóxica no fígado, coração e sangue periférico, e os animais tratados com a associação da polpa do maná-cubiu e DXR apresentaram uma redução de danos ao DNA. Nas doses de 250, 375 e 500 mg/kg p.c., a polpa do maná-cubiu foi capaz de reduzir a peroxidação lipídica induzida pela DXR em células hepáticas, mas não alterou as concentrações de GSH no fígado e no coração. Os dados obtidos da reação em cadeia da polimerase em tempo real referentes ao gene COX-2 mostraram que a polpa do maná-cubiu não modulou a transcrição deste gene. Ainda, a polpa liofilizada do maná-cubiu apresentou em sua composição a presença de elementos-traço como zinco, manganês, selênio, cobre e crômio, mas não em quantidades significativas. Os compostos bioativos presentes na polpa do maná-cubiu, como carotenoides e compostos fenólicos, podem estar relacionados com os efeitos protetores em certos tecidos e doses de maná-cubiu observados neste estudo. / Cubiuzeiro (Solanum sessiliflorum Dunal) is a native plant from Amazonian Forest, used in folk medicine to treat burns and to control cholesterol and blood glucose levels. Phytochemical analysis of the cubiuzeiro fruit, known as maná-cubiu, revealed that its composition exhibits carotenoids, phenolic compounds and vitamins. Due to the few studies that have assessed the biological activity and the presence of compounds with antioxidant properties in this fruit, the objectives of this study were to evaluate the effects of maná-cubiu pulp on genomic stability, oxidative stress parameters and expression of pro-inflammatory genes (iNos and COX-2) in Wistar rats and to determine the presence of chemical elements in the lyophilized pulp by ICP-MS. The animals received lyophilized maná-cubiu pulp (125, 250, 375 or 500 mg/kg b.w.) by gavage for 14 days. On the last day of treatment, saline or doxorubicin (DXR, 16 mg/kg b.w.), used as a genotoxic agent, were administered intraperitoneally, and after 24 hours the animals were euthanized. Peripheral blood and bone marrow were used in the micronucleus test. In the comet assay, liver, heart and peripheral blood cells were analyzed. Liver and heart tissues were used to analyze the thiobarbituric acid reactive substances and reduced glutathione (GSH) and to evaluate the mRNA expression of COX-2 and iNos genes. The results showed that maná-cubiu pulp was not mutagenic or cytotoxic in bone marrow and peripheral blood of the animals. At 250 and 375 mg/kg b.w. doses, the maná-cubiu pulp reduced the number of micronucleated cells induced by DXR in peripheral blood, while only the 375 mg/kg b.w. dose was antimutagenic in bone marrow cells. The maná-cubiu pulp was not genotoxic on liver, heart and peripheral blood cells, and the animals treated with maná-cubiu and DXR exhibited lower levels of DNA damage. At 250, 375 and 500 mg/kg b.w. doses, maná-cubiu pulp was able to reduce lipidic peroxidation induced by DXR on liver cells, however did not change the GSH levels on heart and liver cells. The data obtained by real time polymerase chain reaction (RT-qPCR) from COX-2 gene showed that maná-cubiu pulp did not alter the expression of this gene. Also, the maná-cubiu pulp presented trace elements as zinc, manganese, selenium, copper and chromium, but not in significant amounts. Bioactive compounds in the maná-cubiu pulp as carotenoids and phenolic compounds may be associated with the protective effect in certain tissues and doses of maná-cubiu shown in this study.

O tratamento tópico do câncer de pele é uma estratégia promissora para aumentar a biodisponibilidade local de antineoplásicos e diminuir efeitos sistêmicos adversos. No entanto, altas concentrações do fármaco nos tumores, que acometem as camadas mais profundas da pele, são requeridas para que o tratamento seja adequado. Para promover a penetração cutânea dos antineoplásicos e atingir o tumor, sistemas de liberação nanoparticulados associados a métodos físicos, como a iontoforese, vêm sendo estudados. Nanopartículas lipídicas sólidas (NLS) são sistemas carreadores explorados para a administração tópica, principalmente, de produtos cosméticos. Pouco se sabe, no entanto, sobre sua influência na penetração cutânea de fármacos e sobre os mecanismos pelos quais as NLS agem para aumentar esta penetração. A iontoforese é um método físico que aumenta a permeação cutânea de fármacos através da aplicação de uma corrente elétrica de baixa densidade. Sua influência na penetração tumoral de fármacos carreados por NLS ainda não foi explorada. Sendo assim, o objetivo deste trabalho foi avaliar a influência da iontoforese na penetração tumoral do antineoplásico modelo doxorrubicina (DOX) a partir de NLS catiônicas. Para tanto, NLS contendo DOX foram preparadas e caracterizadas quanto a distribuição de tamanho, potencial zeta e pH. NLS idênticas, mas marcadas com um fluoróforo lipofílico, o BODIPY FSE-8 (BOD), sintetizado especificamente para este fim, também foram obtidas e caracterizadas. Estas nanopartículas fluorescentes contendo DOX e BOD foram utilizadas para estudar, por microscopia confocal de varredura a laser, in vitro e in vivo, as vias de penetração dos compostos lipofílicos presentes nas NLS e da própria DOX. A penetração da DOX nas diferentes camadas da pele foi avaliada in vitro usando-se células de difusão vertical e pele de suíno. In vivo, a penetração do fármaco foi avaliada também no tumor e no plasma, após 1 h de aplicação passiva e iontoforética das NLS em tumores de células escamosas induzidos em camundongos imunossuprimidos. Nos estudos de microscopia observou-se que a aplicação das NLS levou a uma distribuição mais homogênea da fluorescência no estrato córneo (EC) do que a aplicação de soluções dos fluoróforos livres. A iontoforese aumentou a fluorescência de todas as amostras testadas, levando inclusive a presença de agregados fluorescentes abaixo dos folículos pilosos e a formação de regiões de transporte localizadas mais permeáveis no EC. Nos estudos quantitativos in vitro a iontoforese anódica (a partir do eletrodo positivo) das NLS-DOX levou a concentrações cerca de 39 vezes maiores de DOX na epiderme viável do que todas as outras formulações, indicando um efeito positivo da eletromigração na penetração das NLS catiônicas. Nos estudos in vivo, o aumento da quantidade de DOX acumulada na pele após a iontoforese anódica das NLS-DOX foi bem acentuado frente às outras formulações. Já a presença de fármaco no tumor, apesar de apresentar uma tendência maior de acúmulo quando a iontoforese foi aplicada, não foi estatisticamente diferente das demais formulações. No entanto, a tendência das NLS de ficarem acumuladas na pele, diminuindo a presença da DOX na circulação, foi bastante característica. Pode-se concluir, portanto, que a aplicação de NLS associadas a iontoforese apresenta alto potencial de sucesso para o tratamento tópico, localizado, de tumores cutâneos. / Topical treatment of skin cancer is a promising strategy to increase local bioavailability of antineoplastic drugs and to reduce systemic adverse effects. However, elevated concentrations of the drug in tumors presented in deep skin layers are required for the adequate treatment. To increase drug skin penetration, nanoparticles associated with physical methods, such as iontophoresis, have been studied. Solid lipid nanoparticles (SLN) are drug carrier systems developed for topical administration, especially of cosmetic products. However, almost nothing is known about their influence on the skin penetration of drugs or on the mechanisms by which they enhance drug penetration through the skin. Iontophoresis is a physical method which increases the skin permeation of drugs through the application of a low density electrical current. Its influence on tumor penetration of drugs carried by SLN has not been explored yet. Therefore, the aim of this study was to evaluate the influence of iontophoresis on the penetration of the antineoplastic model drug doxorubicin (DOX) carried by cationic SLN. To this end, SLN containing DOX were prepared and characterized according to their medium size, zeta potential and pH. Besides that, identical SLN containing a lipophilic fluorophore BODIPY FSE-8 (BOD), synthesized specifically for this study, has also been obtained and characterized. These fluorescent nanoparticles containing DOX and BOD were used to study the in vitro and in vivo penetration routes of both DOX and lipophilic compounds present in the SLN, by confocal laser scanning microscopy analysis. The penetration of DOX in the different skin layers was evaluated in vitro using vertical diffusion cells and pig skin. In vivo, the drug penetration was also measured in the tumor and plasma after 1 hour of iontophoretic and passive application of SLN on squamous cells tumors, previously induced in immunosuppressed mice. The microscopy studies showed that the application of SLN resulted in a more homogeneous distribution of fluorescence in the stratum corneum (SC) compared to the application of solutions containing free fluorophores at the same conditions. Iontophoresis increased fluorescence for all samples tested, leading yet to the presence of fluorescent aggregates below the hair follicles and the formation of localized transport regions at the SC. The in vitro quantitative studies showed that anodic iontophoresis (from the positive electrode) of SLNDOX led to about 39 times higher concentrations of DOX in viable epidermis than all the others formulations, indicating a positive effect of electromigration on the penetration of cationic SLN. In the in vivo studies, the amount of DOX accumulated in the skin after anodic iontophoresis of SLN-DOX was also well pronounced. The tendency of SLN accumulation in the skin, reducing the presence of DOX in the blood circulation, was very characteristic. Therefore, it can be concluded that the application of SLN associated with iontophoresis has a great potential for success in the topical treatment of localized skin tumors.