Insights into Mechanisms and Promising Triple Negative Breast Cancer Therapeutic Potential for a Water-Soluble Ruthenium Compound.

Triple negative breast cancer (TNBC) represents a subtype of breast cancer that does not express the three major prognostic receptors of human epidermal growth factor receptor 2 (HER2), progesterone (PR), and estrogen (ER). This limits treatment options and results in a high rate of mortality. We have reported previously on the efficacy of a water-soluble, cationic organometallic compound (Ru-IM) in a TNBC mouse xenograft model with impressive tumor reduction and targeted tumor drug accumulation. Ru-IM inhibits cancer hallmarks such as migration, angiogenesis, and invasion in TNBC cells by a mechanism that generates apoptotic cell death. Ru-IM displays little interaction with DNA and appears to act by a P53-independent pathway. We report here on the mitochondrial alterations caused by Ru-IM treatment and detail the inhibitory properties of Ru-IM in the PI3K/AKT/mTOR pathway in MDA-MB-231 cells. Lastly, we describe the results of an efficacy study of the TNBC xenografted mouse model with Ru-IM and Olaparib monotherapy and combinatory treatments. We find 59% tumor shrinkage with Ru-IM and 65% with the combination. Histopathological analysis confirmed no test-article-related toxicity. Immunohistochemical analysis indicated an inhibition of the angiogenic marker CD31 and increased levels of apoptotic cleaved caspase 3 marker, along with a slight inhibition of p-mTOR. Taken together, the effects of Ru-IM in vitro show similar trends and translation in vivo. Our investigation underscores the therapeutic potential of Ru-IM in addressing the challenges posed by TNBC as evidenced by its robust efficacy in inhibiting key cancer hallmarks, substantial tumor reduction, and minimal systemic toxicity.

MNK1 expression increases during cellular senescence and modulates the subcellular localization of hnRNP A1.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence.

Radiation-induced gastrointestinal (GI) syndrome as a function of age.

Previous studies show increased sensitivity of older mice (28-29 months) compared with young adult mice (3 months, possessing a mature immune system) to radiation-induced GI lethality. Age-dependent lethality was associated with higher levels of apoptotic stem cells in small intestinal crypts that correlated with sphingomyelinase activity, a source of pro-apoptotic ceramide. The objective of this study is to determine whether the cycling crypt base columnar cells (CBCs) in aging animals are specifically more sensitive to radiation effects than the CBCs in young adult mice, and to identify factors that contribute to increased radiosensitivity. Mortality induced by subtotal body radiation was assessed at different doses (13 Gy, 14 Gy, and 15 Gy) in young adult mice versus older mice. Each dose was evaluated for the occurrence of lethal GI syndrome. A higher death rate due to radiation-induced GI syndrome was observed in older mice as compared with young adult mice: 30 vs. 0% at 13 Gy, 90 vs. 40% at 14 Gy, and 100 vs. 60% at 15 Gy. Radiation-induced damage to crypts was determined by measuring crypt regeneration (H&E staining, Ki67 expression), CBC biomarkers (lgr5 and ascl2), premature senescence (SA-ß-gal activity), and apoptosis of CBCs. At all three doses, crypt microcolony survival assays showed that the older mice had fewer regenerating crypts at 3.5 days post-radiation treatment. Furthermore, in the older animals, baseline CBCs numbers per circumference were significantly decreased, correlating with an elevated apoptotic index. Analysis of tissue damage showed an increased number of senescent CBCs per crypt circumference in older mice relative to younger mice, where the latter was not significantly affected by radiation treatment. It is concluded that enhanced sensitivity to radiation-induced GI syndrome and higher mortality in older mice can be attributed to a decreased capacity to regenerate crypts, presumably due to increased apoptosis and senescence of CBCs.

Investigation of the Effects and Mechanisms of Anticancer Action of a Ru(II)-Arene Iminophosphorane Compound in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is one of the breast cancers with poorer prognosis and survival rates. TNBC has a disproportionally high incidence and mortality in women of African descent. We report on the evaluation of Ru-IM (1), a water-soluble organometallic ruthenium compound, in TNBC cell lines derived from patients of European (MDA-MB-231) and African (HCC-1806) ancestry (including IC50 values, cellular and organelle uptake, cell death pathways, cell cycle, effects on migration, invasion, and angiogenesis, a preliminary proteomic analysis, and an NCI 60 cell-line panel screen). 1 was previously found highly efficacious in MDA-MB-231 cells and xenografts, with little systemic toxicity and preferential accumulation in the tumor. We observe a similar profile for this compound in the two cell lines studied, which includes high cytotoxicity, apoptotic behavior and potential antimetastatic and antiangiogenic properties. Cytokine M-CSF, involved in the PI3/AKT pathway, shows protein expression inhibition with exposure to 1. We also demonstrate a p53 independent mechanism of action.

Auranofin-Based Analogues Are Effective Against Clear Cell Renal Carcinoma In Vivo and Display No Significant Systemic Toxicity.

Effective pharmacological treatments for patients with advanced clear cell renal carcinoma (ccRCC) are limited. Bimetallic titanium-gold containing compounds exhibit significant cytotoxicity against ccRCC in vitro and in vivo and inhibit invasion and angiogenisis in vitro and markers driving these phenomena. However, in vivo preclinical evaluations of such compounds have not examined their pharmacokinetics, pathology, and hematology. Here we use NOD.CB17-Prkdc SCID/J mice bearing xenograft ccRCC Caki-1 tumors to evaluate the in vivo efficacies of two titanium-gold compounds Titanocref and Titanofin (based on auranofin analogue scaffolds) accompanied by pharmacokinetic and pathology studies. A therapeutic trial was performed over 21 days at 5 mg/kg/72h of Titanocref and 10 mg/kg/72h of Titanofin tracking changes in tumor size. We observed a significant reduction of 51% and 60%, respectively (p < 0.01) in tumor size in the Titanocref- and Titanofin-treated mice compared to the starting size, while the vehicle-treated mice exhibited a tumor size increase of 138% (p < 0.01). Importantly, no signs of pathological complication as a result of treatment were found. In addition, Titanocref and Titanofin treatment reduced angiogenesis by 38% and 54%, respectively. Microarray and qRT-PCR analysis of ccRCC Caki-1 cells treated with Titanocref revealed that the compound alters apoptosis, JNK MAP kinase, and ROS pathways within 3 h of treatment. We further show activation of apoptosis by Titanocref and Titanofin in vivo by caspase 3 assay. Titanocref is active against additional kidney cancer cells. Titanocref and Titanofin are therefore promising candidates for further evaluation toward clinical application in the treatment of ccRCC.

Patterns of persistent DNA damage associated with sun exposure and the glutathione S-transferase M1 genotype in melanoma patients.

Solar radiation can lead to changes affecting DNA metabolism resulting in loss of DNA integrity. Skin specimens obtained from melanoma patients treated at the Memorial Sloan-Kettering Cancer Center were used to study patterns of DNA fragmentation using the comet assay and levels of deletions in mitochondrial DNA (mtDNA) using real-time PCR. Skin specimens were classified according to the glutathione S-transferase M1 (GSTM1) genotype (either wild type [WT] or null) and patient sunburn history. GSTM1 null individuals with a sunburn history showed increased levels of both DNA fragmentation by comet assays and mtDNA deletions relative to GSTM1 WT patients with little or no sunburn history. Microarray analyses identified a number of genes whose expression was upregulated >or=5-fold in cells from GSTM1-null patients or from those reporting histories of sunburn. These genes encoded small molecule transporters, various growth factor/chemokine receptors, transcription factors and tumor suppressors. Of 17 genes directly involved in DNA repair, three DNA ligases were highly upregulated while the RAD23 UV excision repair gene and the Growth Arrest and DNA Damage gene (GADD45) were downregulated. These findings support the idea that exposure to solar radiation early in life may induce long-term cellular changes that lead to persistent DNA damage and altered patterns of gene expression.

p38 MAP kinase-dependent regulation of the expression level and subcellular distribution of heterogeneous nuclear ribonucleoprotein A1 and its involvement in cellular senescence in normal human fibroblasts.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a RNA binding protein that plays important role in the biogenesis of mRNA, such as alternative splicing and mRNA stability. We have previously demonstrated that hnRNP A1 has diminished protein levels and shows cytoplasmic accumulation in senescent human diploid fibroblasts. Recent reports showed that p38 MAP kinase (p38 MAPK), a member of the MAP kinase family is necessary and sufficient for the cytoplasmic accumulation of hnRNP A1 by stress stimuli such as osmotic shock. p38 MAP kinase has been shown to be involved in cell proliferation and the induction of senescence in response to extracellular stimuli. However, the relationship between hnRNP A1 and p38 MAPK and the roles of hnRNP A1 in cellular senescence have not yet been elucidated. Here we show that hnRNP A1 forms a complex with phospho-p38 MAPK in vivo. Inhibition of p38 MAPK activity with SB203580 elevated hnRNP A1 protein levels and prohibited the cytoplasmic accumulation of the protein, but not hnRNP A2, in senescent cells. The phosphorylation level of hnRNP A1 was elevated in senescent cells. Reduction of hnRNP A1 and A2 levels by siRNA transfection induced a senescence-like morphology and elevated the level of F-actin, a marker of senescence. These results suggest that the expression levels and subcellular distribution of hnRNP A1 are regulated in a p38 MAPK-dependent manner, probably via its phosphorylation. Our results also suggest that hnRNP A2 in addition to hnRNP A1 may play a role in establishing the senescence phenotype.

Chemotherapy-Induced Cognitive Impairment Is Associated with Increased Inflammation and Oxidative Damage in the Hippocampus.

Increasing evidence indicates that chemotherapy results in long-term effects on cognitive dysfunction in some cancer survivors. While many studies have established the domains of cognition and corresponding regions in the brain most affected, little is revealed about the potential molecular mechanisms that mediate these adverse changes after treatment. The effects of chemotherapy on the brain are likely attributed to various mechanisms, including oxidative stress and immune dysregulation, features that are also reminiscent of cognitive aging. We have investigated the cognitive effects of a cocktail composed of doxorubicin and cyclophosphamide (AC-chemo) in a surgical ovariectomized rodent model. In this study, we address whether the levels of pro-inflammatory cytokines and oxidative stress-responsive gene markers are altered in the CNS of rats treated with systemic AC-chemo. We further evaluated the levels of nucleic acids modified by oxidative stress in the hippocampus using both immunohistochemical and Northern blotting techniques with a monoclonal antibody against 8-hydroxyguanosine (8-OHG) and 8-OHdG base lesions. We demonstrate that ERK 1/2 and JNK/SAPK signaling activities are elevated in the hippocampus of AC-chemo rats. The levels of pro-inflammatory, oxidative stress-responsive, and RNA/DNA damage markers were also higher in drug-injected animals relative to saline controls. The results indicate that the effects of AC chemotherapy are associated with oxidative damage and a global stress response in the hippocampus. These alterations in the molecular signature of the brain may underlie the processes that contribute to cognitive impairment after treatment.

Preclinical evaluation of an unconventional ruthenium-gold-based chemotherapeutic: RANCE-1, in clear cell renal cell carcinoma.

BACKGROUND: There are few effective treatments for patients with advanced clear cell renal cell carcinoma (CCRCC). Recent findings indicate that ruthenium-gold containing compounds exhibit significant antitumor efficacy against CCRCC in vitro affecting cell viability as well as angiogenesis and markers driving those 2 phenomena. However, no in vivo preclinical evaluation of this class of compounds has been reported. METHODS: Following the dose-finding pharmacokinetic determination, NOD.CB17-Prkdc SCID/J mice bearing xenograft CCRCC Caki-1 tumors were treated in an intervention trial for 21 days at 10 mg/kg/72h of RANCE-1. At the end of the trial, tumor samples were analyzed for histopathological and changes in protein expression levels were assessed. RESULTS: After 21 days of treatment there was no significant change in tumor size in the RANCE-1-treated mice as compared to the starting size (+3.87%) (P = 0.082) while the vehicle treated mice exhibited a significant tumor size increase (+138%) (P < 0.01). There were no signs of pathological complications as a result of treatment. Significant reduction in the expression of VEGF, PDGF, FGF, EGFR, and HGRF, all key to the proliferation of tumor cells and stromal cells serving protumorigenic purposes was observed. CONCLUSIONS: The tumor growth inhibition displayed and favorable pathology profile of RANCE-1 makes it a promising candidate for further evaluation toward clinical use for the treatment of advanced CCRCC.

Mitochondrial DNA deletions in skin from melanoma patients.

We measured the cellular levels of two types of mitochondrial DNA (mtDNA) deletions--the 4977-bp common deletion and recently identified UVB-induced deletion of 5128 bp--in apparently normal skin obtained from wide excisions in melanoma patients. The number of deleted mtDNAs as well as the total mtDNA copy number was highly variable, but the number of deletions increased with age of the donor almost 12-fold across the age range of the patients. Patients were scored for degree of overall pigmentation and response to sunlight by a phenotypic index (PI). The relative levels of both types of mtDNA deletions were much more abundant in the intermediate PI groups compared with either the low or high PI groups. In the intermediate PI group, melanomas were also seen later in life. Unexpectedly, the complement of total mitochondrial genomes was more than twofold higher in the low PI group than in the other PI groups. This may reflect a proliferative response to DNA damage induced by solar radiation in the high-risk group.

An Approach to Integrating Health Disparities within Undergraduate Biomedical Engineering Education.

Health disparities are preventable differences in the incidence, prevalence and burden of disease among communities targeted by gender, geographic location, ethnicity and/or socio-economic status. While biomedical research has identified partial origin(s) of divergent burden and impact of disease, the innovation needed to eradicate health disparities in the United States requires unique engagement from biomedical engineers. Increasing awareness of the prevalence and consequences of health disparities is particularly attractive to today's undergraduates, who have undauntedly challenged paradigms believed to foster inequality. Here, the Department of Biomedical Engineering at The City College of New York (CCNY) has leveraged its historical mission of access-and-excellence to integrate the study of health disparities into undergraduate BME curricula. This article describes our novel approach in a multiyear study that: (i) Integrated health disparities modules at all levels of the required undergraduate BME curriculum; (ii) Developed opportunities to include impacts of health disparities into undergraduate BME research projects and mentored High School summer STEM training; and (iii) Established health disparities-based challenges as BME capstone design and/or independent entrepreneurship projects. Results illustrate the rising awareness of health disparities among the youngest BMEs-to-be, as well as abundant undergraduate desire to integrate health disparities within BME education and training.

Two-year follow-up of bibliotherapy and individual cognitive therapy for depressed older adults.

This study examined the stability of treatment gains after receiving either cognitive bibliotherapy or individual cognitive psychotherapy for depression in older adults. A 2-year follow-up of 23 participants from Floyd, Scogin, McKendree-Smith, Floyd, and Rokke (2004) was conducted by comparing pre-and posttreatment scores with follow-up scores on the Hamilton Rating Scale for Depression (HRSD) and the Geriatric Depression Scale (GDS). Results indicated that treatment gains from baseline to the 2-year follow-up period were maintained on the HRSD and GDS, and there was not a significant decline from posttreatment to follow-up. There were no significant differences between the treatments on the GDS or HRSD at the 2-year follow-up; however, bibliotherapy participants had significantly more recurrences of depression during the follow-up period.

Spatial Heterogeneity Analysis in Evaluation of Cell Viability and Apoptosis for Colorectal Cancer Cells.

In evaluation of cell viability and apoptosis, spatial heterogeneity is quantified for cancerous cells cultured in 3-D in vitro cell-based assays under the impact of anti-cancer agents. In 48-h experiments using human colorectal cancer cell lines of HCT-116, SW-620, and SW-480, incubated cells are divided into control and drug administered groups, to be grown in matrigel and FOLFOX solution, respectively. Our 3-D cell tracking and data acquisition system guiding an inverted microscope with a digital camera is utilized to capture bright field and fluorescent images of colorectal cancer cells at multiple time points. Identifying the locations of live and dead cells in captured images, spatial point process and Voronoi tessellation methods are applied to extract morphological features of in vitro cell-based assays. For the former method, spatial heterogeneity is quantified with the second-order functions of Poisson point process, whereas the deviation in the area of Voronoi polygons is computed for the latter. With both techniques, the results indicate that the spatial heterogeneity of live cell locations increases as the viability of in in vitro cell cultures decreases. On the other hand, a decrease is observed for the heterogeneity of dead cell locations with the decrease in cell viability. This relationship between morphological features of in vitro cell-based assays and cell viability can be used for drug efficacy measurements and utilized as a biomarker for 3-D in vitro microenvironment assays.

Doxorubicin and cyclophosphamide induce cognitive dysfunction and activate the ERK and AKT signaling pathways.

Chemotherapy is associated with long-term cognitive deficits in breast cancer survivors. Studies suggest that these impairments result in the loss of cognitive reserve and/or induce a premature aging of the brain. This study has been aimed to determine the potential underlying mechanisms that induce cognitive impairments by chemotherapeutic agents commonly used in breast cancer. Intact and ovariectomized (OVX) female rats were treated intravenously with either saline or a combination of cyclophosphamide (40 mg/kg) and doxorubicin (4 mg/kg). All subjects were tested for anxiety, locomotor activity, working, visual and spatial memory consecutively. Although anxiety and visual memory were not affected, chemotherapy significantly decreased locomotor activity and impaired working and spatial memory in female rats, independent of their hormonal status. The cognitive deficits observed are hippocampal dependent. Therefore, as a first step to identity the potential signaling pathways involved in this cognitive dysfunction, the protein levels of extracellular signal-regulated kinase 1/2 (Erk1/2), Akt (neuroprotectant) BDNF and (structural protein) PSD95 in hippocampal lysates were measured. Erk1/2 and Akt pathways are known to modulate synaptic plasticity, neuronal survival, aging and cancer. We found an increased activation of Erk1/2 and Akt as well as an increase in the protein levels of PSD95 in OVX female rodents. However, OVX females had a higher overall BDNF level, independent of chemotherapy. These studies provide additional evidence that commonly used chemotherapeutic agents affect cognitive function and impact synaptic plasticity/aging molecules which may be part of the underlying biology explaining cognitive change and can be potential therapeutic targets.

Mitochondrial deletions in luteinized granulosa cells as a function of age in women undergoing in vitro fertilization.

OBJECTIVE: To test the hypothesis that mitochondrial DNA (mtDNA) deletions are more prevalent in granulosa cells from women of advanced reproductive age than from younger women undergoing IVF. DESIGN: Granulosa cells screened for presence or absence of the 4977-bp deletion in human mtDNA. SETTING: University-based fertility clinic. PATIENT(S): Twenty-four women divided equally between two groups: /=38 years old. INTERVENTION(S): Patients were given gonadotropin stimulation in preparation for IVF with granulosa cells isolated at the time of follicular aspiration. MAIN OUTCOME MEASURE(S): Presence or absence of the 4977-bp deletion in human mtDNA. RESULT(S): Seven out of 12 women analyzed who were <38 years old and 0 out of 12 women who were >38 years old had normal mtDNA as indicated by the presence of the 4977-bp fragment. CONCLUSION(S): These data suggest that women over the age of 38 have granulosa cells that contain a substantial decrease in the level of normal mitochondria as compared with women

Coprologic survey of parasites of spotted hyenas (Crocuta crocuta) in the Masai Mara National Reserve, Kenya.

Seventy fecal samples from spotted hyenas (Crocuta crocuta) in the Masai Mara National Reserve, Kenya were examined for parasite eggs and oocysts using sugar flotation. A total of nine parasite genera were identified, and all samples were positive for at least one parasite species. Most individuals were infected with Ancylostoma sp. and Spirometra sp., and these species had the highest median intensity of infection. Other parasites identified include Isospora sp., Taeniidae, Spirurida, Toxocara sp., Mesocestoides sp., Dipylidium sp., and Trichuris sp.

Competency restoration: an examination of the differences between defendants predicted restorable and not restorable to competency.

According to the U.S. Supreme Court's decision in Jackson v. Indiana (1972), examiners must determine if a defendant has "substantial" probability of regaining competency through treatment "in the foreseeable future." Previous research has indicated that, given the low base rate of defendants unable to be restored to competency, examiners are relatively poor at predicting which defendants will regain competency. Determining the characteristics of not restorable incompetent defendants and restorable incompetent defendants is a necessary first step toward improving examiners' ability to predict a defendant's likelihood of regaining competency. This study examined the competency evaluation reports of 468 defendants evaluated for competency to stand trial. Incompetent defendants significantly differed from competent defendants with regard to age, employment status, ethnicity, criminal charges, and psychiatric diagnosis. Few significant differences existed between defendants predicted restorable and those predicted not restorable by mental health examiners--the differences that did exist were related mainly to nonpsychiatric variables.

Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence.

Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14(ARF) and p16(INK4a). Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14(ARF) isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14(ARF) and p16(INK4a) mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16(INK4a) isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism.

Induction of apoptosis in human replicative senescent fibroblasts.

Cellular senescence is an irreversible growth phase characteristic of normal cells. We have found that human senescent fibroblasts can be induced to undergo programmed cell death (apoptosis) by ceramide, TNF-alpha, or okadaic acid. The most profound effects were induced by TNF-alpha and okadaic acid treatment. In the present study, we also evaluated the contribution of lysosomal activation as a possible mechanism underlying the induction of apoptosis. Four lysosomal enzyme activities were measured: beta-galactosidase, alpha-galactosidase A, beta-glucoronidase, and acid phosphatase. Using an in situ assay, we have found that the activity of beta-galactosidase, which is also a biochemical marker of senescence, is induced in young proliferating fibroblasts following exposure to all three apoptotic inducing agents. The other enzymes were not significantly induced in young fibroblasts following exposure to agents that induce apoptosis. During replicative senescence, three of the four lysosomal enzymes tested (beta-galactosidase, alpha-galactosidase A, and beta-glucoronidase) are constitutively expressed at high levels. TNF-alpha was the only agent that induced lysosomal activity in senescent fibroblasts, of which only alpha-galactosidase A activity was induced. Our studies show that senescent fibroblasts can be induced to undergo apoptosis in a signal-dependent manner. However, the lysosomal enzymes examined do not appear to be correlated with apoptotic induction.