O resveratrol (Res), um polifenol presente no vinho tinto, é conhecido por possuir potente atividade antioxidante. O efeito do resveratrol (Res) frente à nefrotoxicidade do antineoplásico cisplatina (cDDP) foi avaliado em ratos neste estudo. Os animais foram tratados com Res (25 mg/Kg de peso copóreo, ip., dose única) 30 minutos antes da administração de cisplatina (5 mg/Kg de peso copóreo, ip., dose única) e foram sacrificados depois de 2 ou 5 dias do tratamento. Após 5 dias, o aumento da creatinina sérica, volume urinário e proteinúria, que são marcadores de alterações renais, apresentaram significativa redução (p < 0,05) com a administração de resveratrol. Os ratos tratados com cisplatina apresentaram necrose tubular aguda e maior marcação imuno-histoquímica para células ED1 e linfócitos T no córtex e medula externa renal. Estas alterações foram menos intensas nos animais tratados com resveratrol. Após 2 dias, a administração de cisplatina aos ratos induziu aumento na concentração de malonaldeído (MDA) e reduziu nos níveis de glutationa (GSH) no tecido renal, que não foram amenizadas pelo resveratrol. Os resultados desse estudo indicam que o tratamento com resveratrol atenuou as alterações renais funcionais, histológicas e imuno-histoquímicas induzidas pela cisplatina. O efeito protetor provavelmente está relacionado à diminuição de infiltrado de células inflamatórias no tecido renal / Resveratrol (Res), a polyphenolic present in red wine, is known to possess potent antioxidant properties. The ability of resveratrol to protect against the nephrotoxicity of the antineoplastic agent cisplatin (cDDP) was evaluated in rats. The animals were treated with Res (25 mg/Kg body weight, ip., single dose) 30 minutes before administration of cDDP (5 mg/Kg body weight, ip., single dose) and then, sacrificed in 2 or 5 days followed by the treatment. After 5 days with resveratrol administration, the enhanced serum creatinine levels, urinary volume and urinary protein, which are indicative of renal injury, shown a significant reduction (p < 0.05). The cisplatintreated rats presented a tubular cell necrosis and increase immunostaining for ED1 and T-lymphocytes in the renal cortex and outer medulla. Those alterations were less intense in animals treated with resveratrol. After 2 days, administration of cisplatin to rats induced a higher malondialdehyde levels (MDA), and reduction in glutathione (GSH) concentrations in kidney tissue that were not prevented by resveratrol. In this study, the results indicate that resveratrol treatment attenuated the functional, histological and immunohistochemical renal alterations induced by cisplatin. The protect effect is relatated to the decrease of cells infiltrated at kidney tissue.

Cisplatin (CDDP; cis-diamminedichloroplatinum) resistance presents a major impediment in the treatment of several gynecologic solid tumors, including ovarian and cervical tumors. p53, a critical regulator of cellular apoptosis, is a determinant of CDDP sensitivity. In our study, we have observed that the dysregulation of p53 regulators, checkpoint kinase 1 (Chk1) and protein phosphatase magnesium-dependent 1 (PPM1D), significantly reduced CDDP responsiveness in human cancer cells. Isogenic wt-p53 CDDP-sensitive (OV2008) and -resistant (C13*) cervical cancer cells, and isogenic wt-p53 CDDP-sensitive (A2780s) and p53 mutant resistant (A2780cp) ovarian cancer cells, along with CDDP-resistant ovarian cancer cell lines (OCC-1 and OVCAR-3, mutant p53; SKOV-3, p53 null) were used to elucidate the mechanisms of p53 regulation in human gynecologic cancer cells. We have complemented our study with a xenograft model (A2780s) and a tissue microarray of human ovarian tumors to validate our in vitro observations. We have demonstrated that CDDP differentially regulated the p53 activator Chk1 in sensitive and resistant cancer cells; it enhances Chk1 activation in sensitive but not resistant cells. This differential regulation also extended to PPM1D, whereby CDDP enhanced PPM1D content in resistant but not sensitive cells. PPM1D knockdown sensitized resistant cells to CDDP, which was associated with up-regulation of Chk1 and p53 activations, while PPM1D over-expression had the opposite effect. We have also shown that CDDP sensitivity in response to PPM1D down-regulation was p53-dependent. Moreover, CDDP promotes PPM1D nuclear localization in resistant cells and nuclear exclusion in sensitive cells and xenograft tumors. Enhanced PPM1D expression in human ovarian tumors is significantly associated with tumor aggression. Dysregulation of the oncogene Akt has been implicated in a variety of human malignancies, including ovarian cancer. We have demonstrated that Akt regulates PPM1D stability, since activated Akt over-expression in sensitive cells rescued PPM1D from CDDP-induced proteasomal degradation and Akt down-regulation in resistant cells lead to PPM1D de-stabilization and down-regulation. We have shown for the first time that PPM1D is downstream of Akt through which it can modulate CDDP sensitivity in human cancer cells. These findings extend the current knowledge on the molecular basis of CDDP resistance in gynecological cancers and may help in developing effective therapeutic strategies.

Cis-platinum is a widely used cytotoxic agent with known radiosensitising properties. It is used in the treatment of various types of lung cancer that may include radiation to the lung as part of the treatment protocol. There is little evidence and some conflict as to whether it sensitises pulmonary tissue to the effects of radiation treatment. This project investigates the effect of cis-platinum alone or in combination with radiation on mouse lung. Four end points were used to evaluate treatments. They were: the release of pulmonary surfactant, changes in breathing rate, a histology based score of damage and changes in TGF-β - a cytokine important in the development of fibrosis. Single doses of either cis-platinum or radiation, cis-platinum given immediately before a single dose of radiation, cis-platinum given immediately before the first of two fractions of radiation and cis-platinum given at various times before and after a single dose of radiation were investigated. Cis-platinum alone was observed to cause an increase in the phospholipid content of lavaged surfactant. Cis-platinum was observed to cause an early release in surfactant and a trend existed for it to induce an early increase in breathing rates as compared to that induced by radiation alone. Cis-platinum was observed to increase radiation damage as assessed using a histology based scoring system. Higher TGF-β levels in lavaged surfactant were observed in C57 /Bl mice as compared to Balb/C. No difference in TGF-β levels was seen in homogenised lung between the strains. Cis-platinum may cause changes in TGF-β in C57/Bl mice but further work is necessary to confirm this.

Indiana University-Purdue University Indianapolis (IUPUI) / Testicular cancer is the most common solid tumor among young men. Although testicular cancer survivors (TCS) are expected to live for over 40 years after cancer diagnosis, they are at risk for chemotherapy adverse effects such as hearing loss (HL), tinnitus, and psychosocial effects. The aim of this study was to investigate factors associated with discrepancies between subjective and objective HL, factors associated with HL, and factors associated with the use of medications for anxiety/depression. TCS were enrolled in the Platinum Study. Sociodemographic characteristics, health behaviors, morbidities, and prescription medications were assessed though self-reporting using validated questionnaires. Bilateral pure-tone air conduction thresholds were collected at frequencies 0.25-12 kHz. To assess HL severity, hearing thresholds were classified according to American Speech-Language-Hearing Association criteria. Multivariable multinomial, ordinal, and binomial logistic regressions were used to test factors for association with discrepancy between subjective and objective HL, cisplatin-induced HL, and use of medications for anxiety/depression, respectively. Patients with HL at only extended high frequencies (10-12 kHz) could perceive hearing deficits; thus, it would be preferable for these frequencies to be included in audiometric assessments of cisplatin-treated adult-onset cancer survivors. Age, no noise exposure, and mixed/conductive HL were significantly associated with more underestimation of HL severity. Hearing aid use and education were significantly associated with less underestimation of HL severity. Having tinnitus was associated with more overestimation of HL severity. Age, cumulative cisplatin dose, and hypertension showed significant association with greater HL severity, whereas post-graduate education was associated with less HL severity. Factors associated with more use of medications for anxiety/depression were tinnitus, and peripheral sensory neuropathy, while being employed and engaging in physical activity were significantly associated with less use of anxiety/depression medications. The sole use of patient-reported measures of HL might not be well-suited to evaluate HL in cancer survivors; thus, the use of audiometry may complement patient-reported HL. In terms of modifiable risk factors of cisplatin-induced HL, healthcare providers should monitor patients’ blood pressure and manage hypertension appropriately. In addition, healthcare providers need to effectively manage tinnitus and peripheral neuropathy to improve treatment outcomes of anxiety and depression. / 2022-05-19

Regulators of G-protein signaling (RGS) are a multifunctional and highly diverse group of proteins that negatively regulate G-protein coupled receptor (GPCR) signaling pathways. The common mechanism of RGS is to act as GTPase accelerating proteins (GAP) to accelerate the hydrolysis of active GTP bound G proteins and terminate the actions of the associated GPCR. In addition to the traditional function of inhibiting G-protein signaling, recent studies have highlighted the role of RGS proteins in modulating GPCRs in GAP-independent way. There are more than 30 RGS proteins, and depending upon cell/tissue type, they interact and associate with different G proteins and GPCRs to modulate various physiological functions. RGS17, a member of the RGS-RZ subfamily, commonly targets GTP bound Gαz, Gαi, and Gαo for hydrolysis and signal termination. RGS17 is abundantly expressed in the central nervous system and is highly associated with opioid, dopamine and cannabinoid receptors in the brain. RGS17 is also upregulated in many malignant tumors such as lung, prostate and breast cancers. Analysis of whole cochlea transcriptome data from our lab revealed higher levels of RGS17 in the cochlea after cisplatin treatment. This highlights a possible role of RGS17 (and probably other RGS proteins) in cisplatin ototoxicity. Activation of endogenous, otoprotective GPCRs such as adenosine (A1AR) and cannabinoid (CB2) receptor is beneficial for promoting protection against cisplatin-induced hearing loss. Taking all this together, the underlying hypothesis for this study is that cisplatin could possibly mediate ototoxicity by increasing the expression of RGS17, which reduces the otoprotective effect of endogenous receptors such as cannabinoid receptor 2. The main objective of the study is to examine the expression and function of RGS17 in the cochlea and determine if inhibition of RGS17 could protect against cisplatin ototoxicity.The expression of RGS17 was observed in the both in vitro and in vivo models of cochlear cell types. Immunofluorescence study, western blot analysis, and RT-qPCR results showed the presence of RGS17 in UB\OC-1, as well as Wistar rat cochlea; expression levels increased after cisplatin treatment. To determine the role of RGS17 in hearing, first, it was overexpressed in the cochlea using adenoviral vector that was found to significantly increase ABR threshold shifts and decrease ABR Wave I amplitude. Conversely, knockdown of RGS17 (by siRGS17) decreased cisplatin-induced elevations in ABR thresholds along with increased wave I amplitude and latency. Furthermore, siRGS17 pretreatment prevented cisplatin-mediated synapse loss at inner hair cells. This indicates inhibition of RGS17 can preserve the functional and physiological integrity of the cochlea, which is essential for hearing. Cochleae that were treated with siRGS17, followed by cisplatin, showed fewer TUNEL-positive cells and reduced loss of Outer hair cells (OHC) as compared to cisplatin-treated rats. Moreover, overexpression of RGS17 increased the ratio of the transcription factors, pSTAT1/pSTAT3, which may indicate initiation of the apoptotic pathway. Moreover, UB\OC-1 cells treated with Celastrol, a RGS17 inhibitor, showed an increase in cell viability against cisplatin toxicity. In addition to apoptosis, overexpression of RGS17 also elevated ROS production and oxidative stress. But, the inhibition of RGS17 attenuated the cisplatin-induced increase in transcripts for oxidative and inflammatory stress markers, such as NOX3, iNOS, KIM1, TNF-α, and COX2, whereas the mRNA level of antioxidant genes such as Nrf2 and SOD2 were increased. Activation of CB2 via JWH-015 (a CB2 agonist) prior to cisplatin administration significantly reduced the cisplatin-induced elevated levels of RGS17, while knockdown of CB2 increased RGS17 expression in the cochlea. siRGS17 treatment boosted endogenous CB2R-Gα expression. Additionally, cisplatin decreased the expression of Gαi/o and Gαz in vitro, but the activation of CB2 increased the expression of these G proteins. Furthermore, JWH-015 treatment alleviated RGS17-dependent cell death. This study suggests that RGS17 could serve as a mediator of cisplatin ototoxicity by reducing the duration of active CB2R-G protein signaling, which normally suppresses cochlear oxidative stress, inflammation and hair cell apoptosis, and thereby preserves normal hearing. These data also indicate the existence of tonic reciprocal inhibition between RGS17 and CB2 mediated via the G proteins. Thus, we propose that RGS17 inhibitors could serve as an effective treatment against cisplatin ototoxicity when used alone and can potentiate the actions of CB2 agonists when used in combination therapy against cisplatin-induced hearing loss.

No description available.

No description available.

The repair of DNA damage is vital to the health and survival of organisms and their cells. In humans, there exist several disorders that involve the inefficient processing or repair of DNA damage. Cellular sensitivity to DNA-damaging agents is a hallmark of repair-deficient syndromes which are often associated with an increased risk of cancer. In this work, I have investigated the repair of cisplatin-damaged DNA by utilizing host cell reactivation and cellular capacity assays that assess DNA repair using adenovirus (Ad) as a probe. Cisplatin is a widely used chemotherapeutic drug that induces both intrastrand and interstrand crosslinks in DNA. The host cell reactivation (HCR) assay examines the ability) of host cells to repair and hence, replicate cisplatin-damaged Ad DNA. This assay is believed to primarily be a measure of bulk nucleotide excision DNA repair. The cellular capacity assay examines the ability of cisplatin-damaged cells to support the replication of undamaged Ad DNA, and is thought to reflect the repair of the active cellular genes necessary for Ad replication. The repair of cisplatin-damaged DNA was studied in three human genetic syndromes -Roberts syndrome (RS), xeroderma pigmentosum (XP) and Li-Fraumeni syndrome (LFS). Fanconi's anemia (FA) cells were also used as a contml strain. RS is characterized by growth retardation, limb reductions and craniofacial abnormalities. Cell; from a subset of RS patients, termed RS+, are hypersensitive to several DNA-damaging agents, and it has been suggested that this hypersensitivity may result from a deficiency in the DNA repair capacity of these cells. (XP patients are sensitive to ultra violet light and are prone to the development of skin cancers. Cells from these patients are deficient in the nucleotide excision repair (NER) pathway responsible for repair of UV -induced lesions.) Patients with FA have a variety of congenital abnormalities, including a high susceptibility to leukemia. FA cells are sensitive to DNAcrosslinking agents such as mitomycin C (MMC) and cisplatin. Using the HCR and cellular capacity assays, deficiencies in DNA repair were detected in the XP and FA fibroblasts but not in the RS+ fibroblasts when compared to normal strains. The NERdeficient XP cells showed a significant reduction in both HCR of cisplatin-damaged Ad and in their capacity to support Ad replication following cellular cisplatin damage suggesting that cisplatin damage is repaired at least in part by the NER pathway. The normal HCR and capacity response of the RS+ cells compared to the XP cells suggests that the hypersensitivity of RS+ cells to DNA damage is not due to a deficiency in NER. The FA cells had normal HCR of cisplatin-damaged Ad but were significantly reduced in their capacity to support Ad replication following cisplatin treatment which was attributed to a defici,~ncy in the repair of DNA interstrand crosslinks. RS+ cells were not reduced in their capc.city to support Ad DNA replication, suggesting that the RS+ cellular hypersensitivity doe; not result from a deficiency in interstrand crosslink repair as seen with FA cells. LFS is a cancer prone syndrome that involves mutations in the p53 tumour suppressor gene. It was found that HCR of cisplatin-damaged Ad was normal in both p53-heterozygous and -hemizygous LFS cells, whereas the NER-deficient XP cells had significantly reduced HCR. The capacity of cisplatin-damaged, p53-heterozygous LFS fibroblasts was significantly reduced compared to normal cells. This suggests that although the LFS fibroblasts appear to have normal bulk NER, as shown by HCR, they appear to be deficier in the repair of the actively transcribed cellular genes necessary for viral replication. These results suggest a role for p53 in the repair of cisplatin damage of active genes. / Thesis / Master of Science (MS)

It is has been observed in several cell systems that cisplatin can radiosensitize and that the response of cells to combination cisplatin and radiation depends on several factors. These include the radiation dose and drug concentration used, the order in which the two treatments are administered, and the time between their administration. The response of a head and neck squamous carcinoma cell line to combination cisplatin-radiation treatment was examined. The response was found to be additive when cisplatin was given first, regardless of the timing and magnitude of the treatments administered. When cells were treated with radiation first, antagonism was observed for low radiation doses and drug concentrations. The response may be explained by a low radiation dose induction of processes that protect the cell from a second damaging agent, similar to the adaptive response. There is some indication in the literature that cisplatin can preferentially radiosensitize cells that are proficient in certain types of DNA repair. Therefore, the response of a cisplatin-resistant strain of the SCC-25 cell line was also investigated. The cisplatin-resistant cell line was found to be substantially radiosensitized by cisplatin for moderate amounts of radiation and cisplatin. The results are discussed with reference to the current proposed mechanisms for cisplatin-radiation interaction. / Thesis / Master of Science (MS)

The goal of this project was to design a new kind of tagged supramolecular structural motif. These systems are modular in design and able to bind to DNA. The motif can be represented as TAG-LA-BL-BAS (TAG = NMR active phosphine ligand, LA = light absorber, BL = bridging ligand and BAS = bioactive site). The TAG provides a NMR probe for the characterization of supramolecular complexes as well as for the future investigation of the metal complex−DNA interaction process. In this project the phosphorous ligand PEt₂Ph was selected as the TAG due its ability to provide an easy ³¹P NMR probe in the research. The LA represents the light absorber which could be photoexcited by photons of proper energy, here a Ru^II(tpy)(PEt₂Ph)(BL) chromophore is used. The bridging ligands are those bidentate polyazine ligands that can connect two metal center together in a polymetallic system and have a low energy π* orbital. The BAS represents the bioactive sites for binding to DNA, in this case the cis-Pt^IICl₂ moiety based on previous studies with cisplatin. The Ru-Pt bimetallic complexes [(tpy)Ru(PEt₂Ph)(BL)PtCl₂](PF₆)₂ (BL = bpm or dpp) and their precursors were designed, successfully synthesized and characterized. The synthesis followed a building block approach, allowing variation of the supramolecular system. The final bimetallic complexes were made without need for Al₂O₃ column chromatograph, important due to the presence of the labile P^IICl₂ center. The bimetallic complexes and all of their monometallic precursors were fully characterized by FAB MS, electrochemistry, electronic absorption and ³¹P NMR spectroscopy. The Ru-Pt bimetallic complex containing the bpm bridging ligand and its precursors were also characterized by ¹H NMR. The FAB MS spectra of the complexes is characterized by the appearance of the parent ion peaks [M-PF₆]⁺ and [M-2PF₆]⁺ . The cyclic voltammogram of all complexes show metal based oxidation(s) and ligand based reductions. The electronic absorption spectra of the complexes are characteristic of the lowest lying Ru to bridging ligand MLCT (metal to ligand charge transfer) transition with higher energy bridging and terminal ligand based Pi to Pi* transitions. The electronic absorption data are consistent with the electrochemical data. The ³¹P NMR technique provides an efficient and easy characterization method for the complexes, showing the utility of this structural moiety. The DNA binding activity of the bimetallic complexes were studied by non-denaturing agarose gel electrophoresis and the results show that these tagged bimetallic complexes can bind to DNA through the cis-Pt^IICl₂ moiety. This binding has a more pronounced retardation effect on DNA migration than cis-[Pt(NH₃)₂Cl₂] (cisplatin), but less than [Ru(bpy)₂(dpq)PtCl₂](CF₃SO₃)₂. The DNA binding study establishes these bimetallic complexes with a NMR tag ligand, PEt₂Ph, as a new kind of DNA binding agent. / Master of Science