Sergio Granados

Periodontitis is associated with altered plasma fatty acids and cardiovascular risk markers.

BACKGROUND AND AIMS In periodontitis it has been found that some perturbation exists in lipid biomarkers, such as increased serum total cholesterol and low-density lipoprotein cholesterol. Nevertheless, the relationship between fatty acids and periodontitis has been demonstrated only in a few studies and remains controversial. The aim of this investigation was to explore the effects of periodontitis on a cluster of traditional and novel cardiovascular risk factors such as plasma-lipids profile, types of plasma fatty acids, adhesion molecules and systemic inflammatory markers. METHODS AND RESULTS At a university dental school, 56 patients all over 35 years old were enrolled and invited to participate in the study. Total plasma fatty acids, saturated, n-6 polyunsaturated and monounsaturated fatty acids, peroxidability index, soluble VCAM, TNF-alpha, cholesterol, triacylglycerols, and VLDL-c were significantly higher in the periodontitis group compared to the non-periodontitis group. CONCLUSIONS This close association found between plasma triacylglycerols, LDL-c, saturated fatty acids, polyunsaturated fatty acids, total amount of fatty acids and coenzyme Q(10) with some periodontal data such as periodontal probing depth, recession of the gingival margin and clinical attachment level (Pearson correlation between 0.3 and 0.6), leads to the conclusion that there is an inter-relationship between periodontitis, plasma fatty acids profile and the increase in metabolic risk factors for cardiovascular diseases.

Curcumin ameliorates rabbits's steatohepatitis via respiratory chain, oxidative stress, and TNF-α

The pathophysiology of nonalcoholic steatohepatitis (NASH) is still not fully understood, and available treatments are not entirely satisfactory. Steatosis progression to NASH is associated with deleterious action of reactive oxygen species, mitochondrial dysfunction, and inflammatory cytokines. We investigated the use of curcumin (compared to vitamin E) in the treatment of NASH. Experimental NASH was induced in rabbits by the intake of a high-fat diet. Oxidative stress status, histology, lipid metabolism, and TNF-alpha protein levels were assessed in liver. The high-fat diet induced pathologically assessed NASH, and compared to healthy controls, raised the levels of aminotransferases, reduced mitochondrial antioxidants, increased mitochondrial reactive oxygen species, and led to poor mitochondrial function as well as to higher TNF-alpha protein levels. Curcumin administration together with the high-fat diet led to rabbits with a lower NASH grade and lower levels of aminotransferases, higher values for mitochondrial antioxidants, lower mitochondrial reactive oxygen species, an improved mitochondrial function, and lower levels of TNF-alpha protein levels. Vitamin E treatment was unable to reduce NASH. In conclusion, curcumin might be useful in the management of NASH through a mechanism involving the antioxidant, anti-inflammatory, and mitochondrial-protective potential of curcumin.

Chloroquine eliminates cancer stem cells through deregulation of Jak2 and DNMT1.

Triple negative breast cancer (TNBC) is known to contain a high percentage of CD44+/CD24−/low cancer stem cells (CSCs), corresponding with a poor prognosis despite systemic chemotherapy. Chloroquine (CQ), an antimalarial drug, is a lysotropic reagent which inhibits autophagy. CQ was identified as a potential CSC inhibitor through in silico gene expression signature analysis of the CD44+/CD24−/low CSC population. Autophagy plays a critical role in adaptation to stress conditions in cancer cells, and is related with drug resistance and CSC maintenance. Thus, the objectives of this study were to examine the potential enhanced efficacy arising from addition of CQ to standard chemotherapy (paclitaxel) in TNBC and to identify the mechanism by which CQ eliminates CSCs in TNBCs. Herein, we report that CQ sensitizes TNBC cells to paclitaxel through inhibition of autophagy and reduces the CD44+/CD24−/low CSC population in both preclinical and clinical settings. Also, we are the first to report a mechanism by which CQ regulates the CSCs in TNBC through inhibition of the Janus‐activated kinase 2 (Jak2)—signal transducer and activator of transcription 3 signaling pathway by reducing the expression of Jak2 and DNA methyltransferase 1. Stem Cells 2014;32:2309–2323

Influence of nutritional iron deficiency anemia on DNA stability and lipid peroxidation in rats

OBJECTIVE Oxidative stress results from an imbalance between the formation and neutralization of pro-oxidants. Disturbance of the pro-oxidant/antioxidant balance is also considered to be a causative factor underlying oxidative damage to cellular molecules, such as DNA, causing strand breaks. There is considerable controversy about the antioxidant status in iron-deficiency anemia (IDA), but scant information is available regarding DNA integrity. In the present study, we investigated the relation between DNA stability and hepatic antioxidant capacity in rats with induced IDA. METHODS Peripheral DNA damage was assessed using an alkaline comet assay. Further, the hepatic antioxidant enzyme glutathione peroxidase and the production of thiobarbituric acid-reactive substances were measured in control rats and in those with induced IDA. RESULTS Comparison of the control and anemic rats showed no differences in thiobarbituric acid-reactive substances production in the cytosolic fraction of hepatic cells. Nor were there any differences in liver glutathione peroxidase enzyme activity or DNA stability, as demonstrated by the percentage of DNA in the head (90.77 in control rats versus 88.23 in anemic rats), tail (9.23 in control rats versus 11.76 in anemic rats), and olive tail moment (0.155 in control rats versus 0.141 in anemic rats). CONCLUSION IDA does not affect DNA stability or lipid peroxidation in rats, suggesting that there is enough compensatory capacity to keep antioxidant defenses high.

Role of RPL39 in Metaplastic Breast Cancer.

Abstract Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer. Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39. Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = .006) and iNOS expression (P = .003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor NG-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = .04 and P = .02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1. Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer.

Hydroxytyrosol as a Component of the Mediterranean Diet and Its Role in Disease Prevention

Hydroxytyrosol is a polyphenol that forms part of the minor compound fraction of extra virgin olive oil, one of the most important elements in the Mediterranean diet. Because of the low incidence of different diseases in Mediterranean countries, such as cardiovascular diseases and cancer, plenty of studies have been performed showing which components of this healthy diet are responsible for these beneficial effects, and most of them have reported that hydroxytyrosol is one of these components. Because of the nature of this element, many studies of this isolated component have been performed using different hydroxytyrosol-enriched olive oils. This research has demonstrated hydroxytyrosol’s potential as an antioxidant, anti-inflammatory, and antiatherogenic agent and its role in the prevention of different diseases, together with some other direct activities such as antitumor action or as an inhibitor of the expression of different cellular receptors. In addition, a relation with the expression of some specific proteins involved in many diseases has been documented in the scientific literature. Other studies have focused on its antimicrobial and dermatological activities and its importance in healing and epithelization after surgery. All these findings allow a possible role as a nutraceutical in the prevention and treatment of some pathologies to be postulated.

Abstract 3495: Tumor growth arrest induced by a proprietary inhibitor of JAK2 on TNBC patient-derived xenografts

Introduction: Triple-negative breast cancer (TNBC) has an increased risk for metastasis and poor long-term survival. Breast Cancer Stem Cells (BCSC) are a subpopulation of treatment-resistant cells that survive and re-initiate tumor growth and seed metastases. A specific and proprietary JAK2 inhibitor developed by Bristol-Myers Squibb (BMS) is currently in phase 1/2a clinical study in myelofibrosis. We hypothesized that Jak2/STAT3 inhibition may control an inflammatory microenvironment and eliminate BCSCs. The goal of thie study was to run an animal preclinical trail using 10 patient-derived xenograft (PDX) models to determine whether the BMS-inhibitor will target BCSC therapeutically. PDXs resulted from the engraftment of TNBC-patient tumor tissue into immune-deficient mice. They constitute a phenocopy of human tumors; they can predict accurately the effectiveness of novel therapeutics and drug response on patients. Methods: For in vitro, BMS-inhibitor was dissolved in dimethylsulfoxide (DMSO) while 20% citrate/80%, PEG400 was used as vehicle for in vivo studies. In vitro: BT549 and SUM149 TNBC cell lines were treated daily for 72 hours; proliferation was assayed by WST-1. Western blot analysis was used to measure pSTAT3 expression. Wound healing assay were performed to evaluate cell migration. To measure mammospheres forming efficiency (MSFE) cells were seeded in Methylcellulose-Based Media. In vivo: for each PDX model, treatments included: vehicle, BMS-inhibitor, docetaxel, and docetaxel + BMS-inhibitor. Animals received three cycles of treatment, 14 days each; docetaxel was delivered at day one; BMS-inhibitor was administered daily for five days, followed by two days of “drug holiday”. Tumor volume was measured twice weekly. In the present abstract we report the results of 4 chemotherapy resistant PDXs which include BCM-5998, BCM-4272, BCM-2147 and BCM-3107 models. Results: BMS-inhibitor impaired cell proliferation, declined pSTAT3 levels and reduced cell migration. In BT549 cells, it lowered MSFE of both primary and secondary mammospheres. BMS-inhibitor increased docetaxel cell toxicity when given together. In vivo, this inhibitor alone reduced BCM-5898 tumor growth when compared to vehicle. Combination therapy (i.e., BMS- inhibitor /docetaxel) induced tumor growth arrest and improved survival rate in all models tested. Conclusions: BMS-inhibitor decreased cell proliferation and capacity to migrate and to form mammospheres; it sensitized TNBC PDXs to docetaxel, resulting in tumor growth arrest and improved survival rate. The BMS-inhibitor/docetaxel combination therapy warrants additional studies as it shows promising therapeutic efficacy and improved survival rates in TNBC models. Furthermore, we speculate that including additional inhibitors to target other BCSC signature pathways, would possibly improve chemotherapy action and lead to complete tumor regression. Citation Format: Daniel Davila-Gonzalez, Sergio Granados, Roberto Rosato, Jenny C. Chang. Tumor growth arrest induced by a proprietary inhibitor of JAK2 on TNBC patient-derived xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3495. doi:10.1158/1538-7445.AM2015-3495

Abstract 4024: iNOS inhibition increases survival in triple negative breast cancer by targeting metastasis and epithelial-mesenchymal transition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in breast cancer patients by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. We also propose an effective clinically relevant-targeted therapeutic regimen by re-purposing the iNOS inhibitor L-NMMA which has been extensively investigated in thousands of patients with cardiogenic shock. Cell migration was determined in confluent MDA-MB-231 cells with a “wound healing assay” treated with different concentrations of the specific iNOS inhibitor 1400W (0, 0.0001, 0.001, 0.01, 0.1, 1, 2, 4 mM) in low serum conditions (1%) for 72h. EMT-inducing factors (Snail, Slug, Twist1, and Zeb1) were determined by Western blot in MDA-MB-231 cells. For in vivo studies, 3x106 MDA-MB-231 and SUM159 cells were injected in the right mammary fat pad of female SCID Beige mice (n=10/group). The clinically relevant dose regimen consisted on two cycles of docetaxel (20mg/kg, i.p, on day 0) twelve hours before being combined with L-NMMA (400mg/kg on day 1, and 200mg/kg for 4 additional days by oral gavage) and amlodipine on day 0 (10mg/kg, i.p, daily, for 6 days). Docetaxel alone, as well as saline (i.p) + sterile water (oral gavage) were used as controls. Our results show that the specific iNOS inhibitor 1400W was able to diminish cell migration in vitro for all concentrations tested. Importantly, impact of iNOS inhibition on cell migration was further correlated with a dose-dependent decrease of the EMT transcription factors- Snail, Slug, and Twist1. Zeb1 was mainly affected at millimolar concentrations. In vivo, the combination of L-NMMA and docetaxel was able to decrease tumor growth in a MDA-MB-231 and SUM159 orthotopic models. Additionally, the combination with amlodipine did significantly prevent the L-NMMA-dependent increase of blood pressure. More importantly, this dose regimen also improved survival compared to docetaxel alone. In conclusion, targeted therapy with iNOS inhibitor impacts cell migration and changes in epithelial-mesenchymal transition might influence metastatic events. Based on these findings, we have planned a phase Ib/II targeted therapeutic trial by re-purposing L-NMMA as an anti-cancer indication. Targeting TNBC with L-NMMA could significantly alter clinical practice for treatment of breast cancer in the near future. Citation Format: Sergio M. Granados, Yi Liu, Bhuvanesh Dave, Melissa D. Landis, Steven S. Gross, Jenny C. Chang. iNOS inhibition increases survival in triple negative breast cancer by targeting metastasis and epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2014-4024

Abstract 2712: Identification of tumor initiating genes RPL39 and MLF2 that mediate lung metastasis through nitric oxide signaling and mesenchymal to epithelial transition.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Triple negative breast cancer (TNBC) is the most aggressive and lethal form of cancer characterized by lack of estrogen, progesterone and Her2 receptors. It is prevalent in women of African American descent, often present in younger and premenopausal women. It shows high risk of recurrence and frequently metastasizes to lungs and brain, resulting in poor overall prognosis. There is no targeted therapy for TNBC as it does not respond to hormonal therapy and is intrinsically resistance to conventional chemotherapy. It is therefore imperative to understand the mechanism of survival of these cancers and unravel its biological pathways and modes of progression. We have previously described breast cancer stem cells (BCSC) to be intrinsically resistant to treatment which is further confirmed by recent publications by other groups that describe direct functional evidence for the same. Using genomic assays, we traced a BCSC gene signature comprising of 477 genes derived from patient biopsies. On selective shRNA knockdown of these genes we identified RPL39 and MLF2 as the top two candidates that affect BCSC self-renewal. Selective siRNA knockdown of RPL39 and MLF2 in human cancer xenografts, showed reduced tumor volume and lung metastases with a concomitant decrease in BCSC markers. Thus, targeting BCSCs in combination with chemotherapy should eliminate the heterogeneous populations within a tumor. Additionally, next generation RNA-seq confirmed mutations in RPL39 and MLF2 in 50% of lung metastases from breast cancer patients. In vitro and in vivo siRNA knockdown of RPL39 and MLF2 showed decrease in nitric oxide synthase, suggesting that these genes are driven by nitric oxide signaling. In conclusion this study reveals novel tumor initiating genes, RPL39 and MLF2 that target the breast cancer stem cells and also show impact on lung metastasis. Our findings enhance the understanding of treatment resistant breast cancer stem cells, the mutations that cause metastases and also lay foundation for developing new therapies for such cancers with poor prognosis. Citation Format: Bhuvanesh Dave, Sergio Granados, Junhua Mai, Dong Soon Choi, Ding Cheng Gao, Sucharita Mitra, Haifa Shen, Senthil Muthuswamy, Vivek Mittal, Mauro Ferrari, Jenny Chang. Identification of tumor initiating genes RPL39 and MLF2 that mediate lung metastasis through nitric oxide signaling and mesenchymal to epithelial transition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2712. doi:10.1158/1538-7445.AM2013-2712

Abstract P5-03-12: Targeting breast cancer stem cells using the autopahgy inhibitor N-Acetyl cysteine

Introduction: One in eight women is diagnosed with breast cancer in the United States. The most aggressive form of breast cancer is the “triple negative breast cancer” (TNBCs), where there is a lack of expression of all three receptors, namely ER, PR and HER2 and a lack of targeted therapies lead to the highest relapse rate in breast cancer. This makes it imperative to identify and target the mechanism of relapse of this cancer. We have recently identified autophagy as a mechanism of tumor resistant cell survival in breast cancer. We have demonstrated an increase in autophagy in chemotherapy treated patients. Further, we have shown that addition of a stem cell inhibitor against NOTCH reduces autophagy and stem cells population. In order to eliminate these tumor resistant cells from surviving chemotherapy, we plan to target the cell survival pathway of autophagy using N-acetyl csyteine as a novel inhibitor. Materials and Methods: We treated three triple negative cell lines (SUM159, BT549 and MDA-MB231) with varying concentrations of N-acetyl cysteine and determined its impact on tumor initiating cells via mammosphere formation and FACS sorting of CD44hi/CD24low cells. N-acetyl cysteine affects mitochondrial metabolism so we tested its impact on mitochondrial DNA mass. Results: N-acetyl cysteine significantly decreased TIC population as evidenced by the remarkable reduction in mammosphere formation efficiency and levels of CD44hi/24low cells at 1 and 10uM in all three cell lines. In two cells lines we have demonstrated that there is a significant increase in mitochondrial mass upon treatment of NAC. Conclusion: We have currently determined that N-acetyl cysteine works via autophagy and eliminates tumor initiating cell population. We have also demonstrated that this involves changing the mitochondrial mass and overall changes in the metabolism of these cells. This novel interlink between mitochondrial metabolism and autophagy provided a new insight into the role of tumor initiating cells in breast cancer and possible new approaches to treat therapy resistance in triple negative breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-12.