Mechanism of synergistic inhibitory effect of benzyl isothiocyanate and zoledronic acid combination on breast cancer induction of osteoclast differentiation.

Bone is the most favored site for metastasis for each major subtype of breast cancer. Therapeutic modalities for alleviation of clinical symptoms associated with bone metastasis include surgical resection, radiation, and bone-targeted therapies, including bisphosphonates (e.g., zoledronic acid; ZA) and a humanized antibody against receptor activator of nuclear factor-κB ligand (denosumab). However, the bone-targeted therapies are expensive, and have poor pharmacokinetic attributes and/or serious adverse effects. Therefore, novel strategies are needed for treatment of bone metastasis or to increase effectiveness of existing bone-targeted therapies. We have shown previously that benzyl isothiocyanate (BITC) is a novel inhibitor of osteoclast differentiation in vitro and bone metastasis in vivo. The present study shows that BITC + ZA combination synergistically inhibits osteoclast differentiation induced by addition of conditioned media from breast cancer cells. These effects were associated with a significant increase in levels of several antiosteoclastogenic cytokines, including interferons, interleukin (IL)-3, IL-4, and IL-27. Kyoto Encyclopedia of Genes and Genomes pathway analysis of RNA-seq data from BITC and/or ZA-treated cells revealed downregulation of genes of many pathways (e.g., actin cytoskeleton, Hippo signaling, etc.) by treatment with BITC + ZA combination, but not by BITC alone or ZA alone. Confocal microscopy confirmed severe disruption of actin cytoskeleton upon treatment of MCF-7 and MDA-MB-231 cells with the BITC + ZA combination. This combination also decreased the nuclear level of yes-associated protein, a core component of Hippo signaling. In conclusion, the present study offers a novel combination for prevention or treatment of bone metastasis of breast cancer.

The Role of MicroRNA-124-3p in Breast Cancer Stem Cell Inhibition by Benzyl Isothiocyanate.

PURPOSE: We have shown previously that benzyl isothiocyanate (BITC) derived from cruciferous vegetables inhibits self-renewal of breast cancer stem-like cells (bCSC). The current study provides insights into the mechanism of bCSC inhibition by BITC. METHODS: Quantitative real time-polymerase chain reaction and western blot analysis were performed to detect microRNAs (miRNAs) and Forkhead box Q1 (FoxQ1) protein expression, respectively. The bCSC were characterized by aldehyde dehydrogenase 1 activity and flow cytometric analysis of CD49f high/CD133high fraction. RESULTS: BITC treatment resulted in induction of miR-124-3p expression in MDA-MB-231 and MCF-7 cells. miR-124-3p did not affect BITC-mediated inhibition of cell migration or cell proliferation but it significantly regulated bCSC in response to BITC. We also found that miR-124-3p directly targets the 3'untranslated regions (UTR) of FoxQ1 and negatively regulates its expression. The BITC-mediated inhibition of bCSC was partially attenuated by miR-124-3p inhibitor. CONCLUSIONS: These findings indicate that miR-124-3p plays an important role in BITC-mediated inhibition of bCSC.

Integrated Automatic Optical Inspection and Image Processing Procedure for Smart Sensing in Production Lines.

An integrated automatic optical inspection (iAOI) system with a procedure was proposed for a printed circuit board (PCB) production line, in which pattern distortions and performance deviations appear with process variations. The iAOI system was demonstrated in a module comprising a camera and lens, showing improved supportiveness for commercially available hardware. The iAOI procedure was realized in a serial workflow of image registration, threshold setting, image gradient, marker alignment, and geometric transformation; furthermore, five operations with numerous functions were prepared for image processing. In addition to the system and procedure, a graphical user interface (GUI) that displays sequential image operation results with analyzed characteristics was established for simplicity. To demonstrate its effectiveness, self-complementary Archimedean spiral antenna (SCASA) samples fabricated via standard PCB fabrication and intentional pattern distortions were demonstrated. The results indicated that, compared with other existing methods, the proposed iAOI system and procedure provide unified and standard operations with efficiency, which result in scientific and unambiguous judgments on pattern quality. Furthermore, we showed that when an appropriate artificial intelligence model is ready, the electromagnetic characteristic projection for SCASAs can be simply obtained through the GUI.

Adjuvant therapy may improve overall survival in high-risk periampullary adenocarcinomas patients - A match-pair analysis from a multi-institutional cohort study (The MIPPAP study).

BACKGROUND: The role of adjuvant therapy in resected periampullary adenocarcinomas is equivocal due to contrasting data and limited prospective trials. METHODS: The Multicentre Indian Pancreatic & Periampullary Adenocarcinoma Project (MIPPAP), included data from 8 institutions across India. Of the 1679 pancreatic resections, 736 patients with T3/T4 and/or Node positive adenocarcinomas (considered as high risk for recurrence) were included for analysis. Three (adjuvant): one (observation) matching, using T3/T4 T staging, nodal positivity and ampullary subtype was performed by using the nearest neighbour matching method. RESULTS: Of 736 patients eligible for inclusion, 621 patients were matched of which 458 patients received adjuvant therapy (AT) (predominantly gemcitabine-based) and 163 patients were observed (O). With a median follow-up of 42 months, there was a statistical difference in overall survival in favour of patients receiving AT as compared to those on observation [68.7 months vs. 61.1 months, Hazard ratio: 0.73 (95% CI: 0.54-0.97); p = 0.03]. Besides AT, presence of nodal involvement (median OS: 65.4 months vs not reached; p = 0.04) predicted for inferior OS. CONCLUSIONS: The results of the match-pair analysis suggest that adjuvant therapy improves overall survival in periampullary adenocarcinomas at high risk of recurrence with a greater benefit in T3/T4, node-positive and ampullary subtypes.

Withaferin A inhibits breast cancer-induced osteoclast differentiation.

Bone is the most prone to metastatic spread of breast cancer cells for each subtype of the disease. Bone metastasis-related complications including severe pain and pathological fractures affect patients' quality of life. Current treatment options including surgery, radiation, and bone-targeted therapies (e.g., bisphosphonates) are costly or have serious adverse effects such as renal toxicity and osteonecrosis of the jaws. Therefore, a safe, inexpensive, and efficacious agent for prevention of breast cancer bone metastasis is urgently needed. Our previously published RNA sequencing analysis revealed that many genes implicated in bone remodeling and breast cancer bone metastasis were significantly downregulated by treatment with withaferin A (WA), which is a promising cancer chemopreventive agent derived from a medicinal plant (Withania somnifera). The present study investigated whether WA inhibits breast cancer induction of osteoclast differentiation. At plasma achievable doses, WA treatment inhibited osteoclast differentiation (osteoclastogenesis) induced by three different subtypes of breast cancer cells (MCF-7, SK-BR-3, and MDA-MB-231). WA and the root extract of W. somnifera were equally effective for inhibition of breast cancer induction of osteoclast differentiation. This inhibition was accompanied by suppression of interleukin (IL)-6, IL-8, and receptor activator of nuclear factor-κB ligand, which are pivotal osteoclastogenic cytokines. The expression of runt-related transcription factor 2, nuclear factor-κB, and SOX9 transcription factors, which positively regulate osteoclastogenesis, was decreased in WA-treated breast cancer cells as revealed by confocal microscopy and/or immunoblotting. Taken together, these data suggest that WA could be a promising agent for prevention of breast cancer-induced bone metastasis.

Forkhead Box Q1 is a novel regulator of autophagy in breast cancer cells.

Forkhead Box Q1 (FoxQ1) transcription factor is overexpressed in luminal-type and basal-type human breast cancers when compared to normal mammary tissue. This transcription factor is best known for its role in promotion of breast cancer stem-like cells and epithelial to mesenchymal transition. The present study documents a novel function of FoxQ1 in breast cancer cells. Overexpression of FoxQ1 in basal-like SUM159 cells and luminal-type MCF-7 cells resulted in increased conversion of microtubule-associated protein light chain 3 beta-I (LC3B-I) to LC3B-II, which is a hallmark of autophagy. Autophagy induction by FoxQ1 overexpression was confirmed by visualization of LC3B puncta as well as by transmission electron microscopy. Expression profiling for genes implicated in autophagy regulation revealed upregulation of many genes, including ATG4B, ATG16L1, CTSS, CXCR4 and so forth but downregulation of BCL2L1, DRAM1, TNF, ULK2 and so forth by FoxQ1 overexpression in SUM159 cells. Western blot analysis confirmed upregulation of ATG4B and CXCR4 proteins by FoxQ1 overexpression in both SUM159 and MCF-7 cells. Chromatin immunoprecipitation assay revealed recruitment of FoxQ1 at the promoter of ATG4B. Pharmacological inhibition of ATG4B using S130 significantly increased apoptosis induction by DOX in empty vector transfected as well as FoxQ1 overexpressing SUM159 and MCF-7 cells but this effect was statistically significantly lowered by FoxQ1 overexpression indicating the protective role of FoxQ1 on apoptosis. Treatment of SUM159 cells with S130 and DOX enhanced LC3B-II level in both empty vector transfected cells and FoxQ1 overexpressing SUM159 cells but not in FoxQ1 overexpressing MCF-7 cells. In conclusion, FoxQ1 is a novel regulator of autophagy.

The Oligostilbene Gnetin H Is a Novel Glycolysis Inhibitor That Regulates Thioredoxin Interacting Protein Expression and Synergizes with OXPHOS Inhibitor in Cancer Cells.

Since aerobic glycolysis was first observed in tumors almost a century ago by Otto Warburg, the field of cancer cell metabolism has sparked the interest of scientists around the world as it might offer new avenues of treatment for malignant cells. Our current study claims the discovery of gnetin H (GH) as a novel glycolysis inhibitor that can decrease metabolic activity and lactic acid synthesis and displays a strong cytostatic effect in melanoma and glioblastoma cells. Compared to most of the other glycolysis inhibitors used in combination with the complex-1 mitochondrial inhibitor phenformin (Phen), GH more potently inhibited cell growth. RNA-Seq with the T98G glioblastoma cell line treated with GH showed more than an 80-fold reduction in thioredoxin interacting protein (TXNIP) expression, indicating that GH has a direct effect on regulating a key gene involved in the homeostasis of cellular glucose. GH in combination with phenformin also substantially enhances the levels of p-AMPK, a marker of metabolic catastrophe. These findings suggest that the concurrent use of the glycolytic inhibitor GH with a complex-1 mitochondrial inhibitor could be used as a powerful tool for inducing metabolic catastrophe in cancer cells and reducing their growth.

The FoxQ1 transcription factor is a novel regulator of electron transport chain complex I subunits in human breast cancer cells.

The FoxQ1 is an oncogenic transcription factor that is overexpressed in basal-like and luminal-type human breast cancers when compared to the normal mammary tissue. The FoxQ1 is implicated in mammary tumor progression. However, the mechanism by which FoxQ1 promotes mammary tumorigenesis is not fully understood. In this study, we present experimental evidence for a novel function of FoxQ1 in the regulation of complex I activity of the electron transport chain. The RNA-seq data from FoxQ1 overexpressing basal-like SUM159 cells revealed a statistically significant increase in the expression of complex I subunits NDUFS1 and NDUFS2 when compared to the empty vector (EV) transfected control cells. Consistent with these results, the basal and ATP-linked oxygen consumption rates were significantly increased by FoxQ1 overexpression in SUM159 and luminal-type MCF-7 cells. The FoxQ1 overexpression in both cell lines resulted in increased intracellular levels of pyruvate, lactate, and ATP that was associated with overexpression of pyruvate dehydrogenase and pyruvate carboxylase proteins. Activity and assembly of complex I were significantly enhanced by FoxQ1 overexpression in SUM159 and MCF-7 cells that correlated with increased mRNA and/or protein levels of complex I subunits NDUFS1, NDUFS2, NDUFV1, and NDUFV2. The chromatin immunoprecipitation assay revealed the recruitment of FoxQ1 at the promoters of both NDUFS1 and NDUFV1. The cell proliferation of SUM159 and MCF-7 cells was increased significantly by overexpression of NDUFS1 as well as NDUFV1 proteins. In conclusion, we propose that increased complex I-linked oxidative phosphorylation is partly responsible for oncogenic role of FoxQ1 at least in human breast cancer cells.

Monocarboxylate transporter 1 is a novel target for breast cancer stem like-cell inhibition by diallyl trisulfide.

Diallyl trisulfide (DATS) is a promising small molecule phytochemical that exhibits in vitro and in vivo activity in multiple preclinical solid tumor models including breast cancer, but the underlying mechanism is not fully understood. We have shown previously that forkhead box Q1 (FoxQ1) transcription factor is a novel target for breast cancer stem-like cells (bCSC) inhibition by DATS. Analysis of the breast TCGA (The Cancer Genome Atlas) data revealed that FoxQ1 expression was positively associated with that of SLC16A1/monocarboxylate transporter 1 (MCT1). Western blot analysis confirmed increased expression of MCT1 protein in SUM159 (basal-like) and MCF-7 cells (luminal-type) stably transfected to overexpress FoxQ1. Furthermore, FoxQ1 was recruited to the promoter of SLC16A1/MCT1. Treatment of SUM159 and MCF-7 cell lines with DATS resulted in suppression of MCT1 protein level that was accompanied by a decrease in intracellular and secreted levels of lactate. Overexpression or knockdown of MCT1 protein failed to alter DATS-mediated inhibition of colony formation or cell migration when compared to corresponding control cells. On the other hand, overexpression of MCT1 protein conferred partial but statistically significant protection against DATS-mediated inhibition of bCSC fraction (CD49fhigh /CD44high and aldehyde dehydrogenase 1 activity). The size of the mammospheres was relatively smaller in the DATS-treated group compared to control group. Inhibition of bCSC upon DATS treatment was augmented by knockdown of the MCT1 protein. In conclusion, the present study reveals that MCT1 is a novel target for bCSC inhibition by DATS treatment.

Histological Spectrum of Clinical Kidney Disease in Type 2 Diabetes Mellitus Patients with special Reference to nonalbuminuric Diabetic Nephropathy: A Kidney Biopsy-based Study.

BACKGROUND: Diabetic nephropathy (DN) is an important and catastrophic complication of diabetes mellitus (DM). Kidney disease has heterogeneity in histology in diabetes patients and includes both diabetic kidney disease (DKD) (albuminuric or nonalbuminuric) and nondiabetic kidney disease (NDKD) either in isolation or in coexistence with DN. Diabetic nephropathy is hard to overturn. While NDKD is treatable and reversible. MATERIALS AND METHODS: We enrolled a total of 50 type 2 diabetes mellitus (T2DM) patients with clinical kidney disease, of both genders and age >18 years, who underwent kidney biopsy from October 2016 to October 2018. Patients with proteinuria <30 mg per day were excluded from the study. The indications of the renal biopsy were nephrotic syndrome (NS), active urinary sediment, rapid decline in renal function, asymptomatic proteinuria, and hematuria. RESULT: A total of 50 (males: 42 and females: eight) patients with T2DM who underwent kidney biopsy were enrolled. The clinical presentation was: NS 26 (52%), chronic kidney disease (CKD) 11 (22%), asymptomatic proteinuria and hematuria six (12%), acute kidney injury (AKI) four (8%), and acute nephritic syndrome (ANS) three (6%). Diabetic retinopathy (DR) was noted in 19 (38%) cases. Kidney biopsy revealed isolated DN, isolated NDKD, and NDKD superimposed on DN in 26 (52%), 14 (28%), and 10 (20%) cases, respectively. Idiopathic membranous nephropathy (MN) (4) and amyloidosis (2) were the most common forms of NDKD, whereas diffuse proliferative glomerulonephritis (DPGN) was the main form of NDKD superimposed on DN. Diabetic nephropathy was observed in 15 (79%) cases in presence of DR and also in 11 (35.5%) cases even in absence of DR. Of eight patients with microalbuminuria four (50%) cases have biopsy-proven DN. CONCLUSION: About 48% of patients had NDKD either in isolation or in coexistence with DN. Diabetic nephropathy was found in absence of DR and in patients with a low level of proteinuria. The level of proteinuria and presence of DR does not help to distinguish DN vs NDKD. Hence, renal biopsy may be useful in selected T2DM patients with clinical kidney disease to diagnose NDKD.

RNA-seq reveals novel cancer-selective and disease subtype-independent mechanistic targets of withaferin A in human breast cancer cells.

Withaferin A (WA) exhibits cancer chemopreventive efficacy in preclinical models representative of two different subtypes of breast cancer. However, the mechanism(s) underlying breast cancer chemoprevention by WA is not fully elucidated. We performed RNA-seq analyses using a non-tumorigenic mammary epithelial cell line (MCF-10A) and human breast cancer cells (BCC) belonging to the luminal-type (MCF-7), HER2-enriched (SK-BR-3), and basal-like subtype (MDA-MB-231) to identify novel cancer-selective mechanistic targets of WA. The WA-regulated transcriptome was strikingly different between MCF-10A versus BCC. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed downregulation of genes associated with cellular senescence in WA-treated BCC. Consequently, the number of senescence-associated ß-galactosidase positive cells was decreased significantly in WA-treated BCC but not in the MCF-10A cells. WA treatment caused upregulation of senescence marker p21 more robustly in BCC than in MCF-10A. Breast cancer prevention by WA in rats was also associated with upregulation of p21 protein expression. The Reactome pathway analyses indicated upregulation of genes associated with cellular response to stress/external stimuli in WA-treated BCC but not in MCF-10A. Two proteins represented in these pathways (HSPA6 and NRF2) were further investigated. While HSPA6 was dispensable for WA-mediated apoptosis and autophagy or inhibition of cell migration, the NRF2 knockout cells were more resistant to apoptosis resulting from WA treatment than control cells. Finally, expression of some glycolysis-related proteins was decreased by WA treatment both in vitro and in vivo. In summary, this study provides novel insights into cancer-selective pathways affected by WA that may contribute to its chemopreventive efficacy in breast cancer.

Outcomes of symptomatic coronavirus disease 19 in maintenance hemodialysis patient in India.

BACKGROUND: Maintenance hemodialysis (MHD) patients face disadvantages with higher risk of acquiring SARS-CoV-2 infection, atypical manifestations, and associated multiple comorbidities. We describe patients' outcomes with symptomatic COVID-19 on MHD in a large cohort of patients from India. METHODS: Data were collected prospectively from hemodialysis units in 11 public and private hospitals between March 15, 2020, and July 31, 2020. The survival determinants were analyzed using stepwise backward elimination cox-regression analysis. RESULTS: Of the 263 total patients (mean age 51.76 ± 13.63 years and males 173) on MHD with symptomatic COVID-19, 35 (13.3%) died. Those who died were older (p = 0.01), had higher frequency of diabetic kidney disease (p = 0.001), comorbidities (p = 0.04), and severe COVID-19 (p = 0.001). Mortality was higher among patients on twice-weekly MHD than thrice-weekly (p = 0.001) and dialysis through central venous catheter (CVC) as compared to arteriovenous fistula (p = 0.001). On multivariate analysis, CVC use (HR 2.53, 95% CI 1.26-5.07, p = 0.009), disease severity (HR = 3.54, 95% CI 1.52-8.26, p = 0.003), and noninvasive ventilatory support (HR 0.59, 95% CI 0.25-0.99, p = 0.049) had significant effect on mortality. CONCLUSION: The adjusted mortality risk of COVID-19 in MHD patients is high in patients associated with severe COVID-19 and patients having CVC as vascular access.

RNA-seq reveals novel mechanistic targets of withaferin A in prostate cancer cells.

Withaferin A (WA) is a promising phytochemical exhibiting in vitro and in vivo anticancer activities against prostate and other cancers, but the mechanism of its action is not fully understood. In this study, we performed RNA-seq analysis using 22Rv1 human prostate cancer cell line to identify mechanistic targets of WA. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the differentially expressed genes showed most significant enrichment of genes associated with metabolism. These results were validated using LNCaP and 22Rv1 human prostate cancer cells and Hi-Myc transgenic mice as models. The intracellular levels of acetyl-CoA, total free fatty acids and neutral lipids were decreased significantly following WA treatment in both cells, which was accompanied by downregulation of mRNA (confirmed by quantitative reverse transcription-polymerase chain reaction) and protein levels of key fatty acid synthesis enzymes, including ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A. Ectopic expression of c-Myc, but not constitutively active Akt, conferred a marked protection against WA-mediated suppression of acetyl-CoA carboxylase 1 and fatty acid synthase protein expression, and clonogenic cell survival. WA was a superior inhibitor of cell proliferation and fatty acid synthesis in comparison with known modulators of fatty acid metabolism including cerulenin and etomoxir. Intraperitoneal WA administration to Hi-Myc transgenic mice (0.1 mg/mouse, three times/week for 5 weeks) also resulted in a significant decrease in circulating levels of total free fatty acids and phospholipids, and expression of ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A proteins in the prostate in vivo.

Myoglobin induces mitochondrial fusion, thereby inhibiting breast cancer cell proliferation.

Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Furthermore, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.

Cytoprotective autophagy induction by withaferin A in prostate cancer cells involves GABARAPL1.

Withaferin A (WA) is a naturally occurring steroidal lactone with proven cancer chemopreventive activity in preclinical models of different cancers including prostate adenocarcinoma. Previously we compared the RNA-seq data from control and WA-treated 22Rv1 human prostate cancer cells to identify mechanistic targets of this phytochemical. The Gene Ontology pathway analysis of the RNA-seq data revealed significant upregulation of genes associated with autophagy upon WA treatment in 22Rv1 cells. In this study, we extended these findings to investigate the mechanism underlying WA-induced autophagy. Initially, we confirmed autophagy induction by WA treatment by transmission electron microscopy using three prostate cancer cell lines (LNCaP, 22Rv1, and PC-3). Fourteen common genes altered by 8- and 16-hour exposure to WA were identified from human autophagy PCR array and these results were consistent with the RNA-seq data. Two key autophagy markers (LC3BII and SQSTM1) were robustly increased in WA-exposed LNCaP, 22Rv1, and PC-3 cells as determined by immunoblotting, and this effect was elevated in the presence of autophagy inhibitor bafilomycin A1 (BafA1). BafA1 treatment augmented WA's cytotoxicity and subsequently its proapoptotic potential. WA treatment induced GABARAPL1 (ATG8L) protein expression in all three cell lines and its knockdown by RNA interference attenuated WA-mediated apoptosis. WA-induced autophagy was not affected in the presence of an antioxidant (EUK134). Taken together, the present study reveals that WA-mediated autophagy is cytoprotective and mediated by GABARAPL1.

Novel mechanistic targets of forkhead box Q1 transcription factor in human breast cancer cells.

The transcription factor forkhead box Q1 (FoxQ1) is overexpressed in different solid tumors including breast cancer, but the mechanism underlying its oncogenic function is still not fully understood. In this study, we compared RNA-seq data from FoxQ1 overexpressing SUM159 cells with that of empty vector-transfected control cells to identify novel mechanistic targets of this transcription factor. Analysis of The Cancer Genome Atlas (TCGA) data set revealed significantly higher expression of FoxQ1 in black breast cancer patients compared with white women with this disease. In contrast, expression of FoxQ1 was comparable in ductal and lobular carcinomas in the breast cancer TCGA data set. Complementing our published findings in basal-like subtype, immunohistochemistry revealed upregulation of FoxQ1 protein in luminal-type human breast cancer tissue microarrays when compared with normal mammary tissues. Many previously reported transcriptional targets of FoxQ1 (eg, E-cadherin, N-cadherin, fibronectin 1, etc) were verified from the RNA-seq analysis. FoxQ1 overexpression resulted in the downregulation of genes associated with cell cycle checkpoints, M phase, and cellular response to stress/external stimuli as evidenced from the Reactome pathway analysis. Consequently, FoxQ1 overexpression resulted in mitotic arrest in basal-like SUM159 and human mammary epithelial cell line, but not in luminal-type MCF-7 cells. Finally, we show for the first time that FoxQ1 is a direct transcriptional regulator of interleukin (IL)-1α, IL-8, and vascular endothelial growth factor in breast cancer cells as evidenced by chromatin immunoprecipitation assay. In conclusion, the present study reports novel mechanistic targets of FoxQ1 in human breast cancer cells.

Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane.

Inhibition of metabolic re-programming represents an attractive approach for prevention of prostate cancer. Studies have implicated increased synthesis of fatty acids or glycolysis in pathogenesis of human prostate cancers. We have shown previously that prostate cancer prevention by sulforaphane (SFN) in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model is associated with inhibition of fatty acid metabolism. This study utilized human prostate cancer cell lines (LNCaP, 22Rv1 and PC-3), two different transgenic mouse models (TRAMP and Hi-Myc) and plasma specimens from a clinical study to explore the glycolysis inhibition potential of SFN. We found that SFN treatment: (i) decreased real-time extracellular acidification rate in LNCaP, but not in PC-3 cell line; (ii) significantly downregulated expression of hexokinase II (HKII), pyruvate kinase M2 and/or lactate dehydrogenase A (LDHA) in vitro in cells and in vivo in neoplastic lesions in the prostate of TRAMP and Hi-Myc mice; and (iii) significantly suppressed glycolysis in prostate of Hi-Myc mice as measured by ex vivo1H magnetic resonance spectroscopy. SFN treatment did not decrease glucose uptake or expression of glucose transporters in cells. Overexpression of c-Myc, but not constitutively active Akt, conferred protection against SFN-mediated downregulation of HKII and LDHA protein expression and suppression of lactate levels. Examination of plasma lactate levels in prostate cancer patients following administration of an SFN-rich broccoli sprout extract failed to show declines in its levels. Additional clinical trials are needed to determine whether SFN treatment can decrease lactate production in human prostate tumors.

Withaferin A inhibits expression of ataxia telangiectasia and Rad3-related kinase and enhances sensitivity of human breast cancer cells to cisplatin.

Ataxia telangiectasia and Rad3-related (ATR) is a serine/threonine-specific kinase that plays an important role in the maintenance of genomic integrity. In this study, we investigated the role of ATR in cell-cycle arrest by withaferin A (WA), a cancer preventative steroidal lactone derived from Withania somnifera plant abundant in India and surrounding countries. The WA treatment decreased the viability of MCF-7, MDA-MB-231, and SUM159 cells. Exposure of breast cancer cells to WA also resulted in suppression of protein level as well as phosphorylation of ATR and its downstream effector kinase (checkpoint kinase 1; CHK1). Both transcriptional and posttranscriptional mechanisms were involved in the WA-mediated downregulation of ATR protein. Downregulation of ATR protein expression resulting from WA exposure was not attenuated by overexpression of manganese superoxide dismutase. In contrast, the overexpression of CHK1 attenuated WA-mediated G2 /M arrest and augmented S10 phosphorylation of histone H3, a marker of mitotic arrest. The protein level of ATR was lowered by about 50% in breast tumors of WA-treated mouse mammary tumor virus-neu mice when compared with vehicle-treated controls but the difference was not significant due to small sample size. WA treatment sensitized MDA-MB-231 and SUM159 cells to growth inhibition and apoptosis induction by cisplatin. Cisplatin treatment resulted in increased autophosphorylation of ATR (T1989) and CHK1 (S345) phosphorylation that was markedly suppressed in the presence of WA. These results indicate that WA is an inhibitor of ATR in human breast cancer cells.

AKT-dependent sugar addiction by benzyl isothiocyanate in breast cancer cells.

The overall promise of breast cancer chemoprevention is exemplified by clinical success of selective estrogen receptor modulators and aromatase inhibitors. Despite clinical efficacy, these interventions have limitations, including rare but serious side effects and lack of activity against estrogen receptor-negative breast cancers. We have shown previously that dietary administration of benzyl isothiocyanate (BITC), which occurs naturally as a thioglucoside conjugate in edible cruciferous vegetables, inhibits development of estrogen receptor-negative breast cancer in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice. This study demonstrates AKT-mediated sugar addiction in breast cancer chemoprevention by BITC. BITC-treated MMTV-neu mice exhibited increased 2-deoxy-2-(18 F)-fluoro-D-glucose (18 F-FDG) uptake in mammary tumors in vivo in comparison with mice fed basal diet. Cellular studies using MDA-MB-231 and SUM159 human breast cancer cell lines revealed BITC-mediated induction and punctate localization of glucose transporter GLUT-1, which was accompanied by an increase in intracellular pyruvate levels. BITC treatment resulted in increased S473 phosphorylation (activation) of AKT in cells in vitro as well as in mammary tumors of MMTV-neu mice in vivo. Increased glucose uptake, punctate pattern of GLUT-1 localization, and intracellular pyruvate levels resulting from BITC exposure were significantly attenuated in the presence of a pharmacological inhibitor of AKT (MK-2206). Inhibition of AKT augmented BITC-mediated inhibition of cell migration and colony formation. BITC-induced apoptotic cell death was also increased by pharmacological inhibition of AKT. These results indicate increased glucose uptake/metabolism by BITC treatment in breast cancer cells suggesting that breast cancer chemoprevention by BITC may be augmented by pharmacological inhibition of AKT.

Etiopathological Study of Crescentic Glomerulonephritis and its Outcome: A Retrospective Analysis.

INTRODUCTION: Crescentic Glomerulonephritis (CGN) is most aggressive structural phenotype and accounts for 2%-7% of renal biopsy in most series. The aim of study was to assess the clinical feature and outcome of CGN at our centre. MATERIAL AND METHODS: The renal biopsy performed during the period of January 2015 to January 2018 was studied and patients showing crescentic glomerulonephritis on histology were selected for this study. The clinical presentation, immunological assay, biochemical and haematological investigations, treatment protocol and final outcome at three month of these patients were analysed in the present study. RESULTS: Of 380 biopsy, 26 (male=17, female=9) patients had histological evidence of CGN (6.8%). The age of patients ranged between 13-75 (mean=43) years. Fibro cellular and cellular crescent was noted in 84.61% and 15.38% of patients respectively. Small vessels vasculitis and granuloma was observed in 5 (19.23%) cases. Based on immunohistopathology, we observed type I (n=3), type II (n=8), type III (n=5), type IV (n=3), and type V (n=7) crescentic GN in 11.53%, 30.76%, 19.23%, 11.53% and 26.92% of patients respectively. Haemodialysis was given to 22(84.61%) and 4(15.38%) patients were treated with immunosuppressive therapy. Plasmapheresis was used in two double positive (ANCA + Anti GBM Ab) patients. Remaining 21(80.76%) has progressed to ESRD over a period of 2-3 months. CONCLUSION: Type II (immune complex) CGN was most common type followed by type V (immune negative) and type III (pauci-immune) CGN. The crescentic GN had worse prognosis with >80% of patients progressed to ESRD within 3 month of time from onset of illness. Early diagnosis and treatment is associated with favourable outcome.