Modulation of the oral glucocorticoid system during black raspberry mediated oral cancer chemoprevention.

Recent reports suggest that glucocorticoids (GCs), which can be synthesized in the oral mucosa, play an important role in cancer development. Therefore, the objectives of this study were to characterize the role of the oral GC system in oral cancer, and determine the effect of black raspberry (BRB) administration on GC modulation during oral cancer chemoprevention. We determined the expression of GC enzymes in various oral cancer cell lines, and investigated the role of the GC inactivating enzyme HSD11B2 on CAL27 oral cancer cells using siRNA mediated knockdown approaches. Using two in vivo models of oral carcinogenesis with 4-nitroquinoline 1-oxide carcinogen on C57Bl/6 mice and F344 rats, we determined the effect of BRB on GC modulation during head and neck squamous cell carcinoma chemoprevention. Our results demonstrate that HSD11B2, which inactivates cortisol to cortisone, is downregulated during oral carcinogenesis in clinical and experimental models. Knockdown of HSD11B2 in oral cancer cells promotes cellular proliferation, invasion and expression of angiogenic biomarkers EGFR and VEGFA. An ethanol extract of BRB increased HSD11B2 expression on oral cancer cells. Dietary administration of 5% BRB increased Hsd11b2 gene and protein expression and reduced the active GC, corticosterone, in cancer-induced mouse tongues. Our results demonstrate that the oral GC system is modulated during oral carcinogenesis, and BRB administration upregulates Hsd11b2 during oral cancer chemoprevention. In conclusion, our findings challenge the use of synthetic GCs in head and neck cancer, and support the use of natural product alternatives that potentially modulate GC metabolism in a manner that supports oral cancer chemoprevention.

STAT1 is regulated by TRIM24 and promotes immunosuppression in head and neck squamous carcinoma cells, but enhances T cell antitumour immunity in the tumour microenvironment.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a significant problem and is frequently resistant to current treatments. STAT1 is important in anti-tumour immune responses against HNSCC. However, the role of STAT1 expression by tumour cells and its regulation during HNSCC is unclear. METHODS: We determined the effects of STAT1 inhibition on tumour development and immunity in CAL27 and UMSCC22A HNSCC cell lines in vitro and in a HNSCC carcinogen-induced model in vivo. RESULTS: STAT1 siRNA knockdown in human HNSCC cells impaired their proliferation and expression of the immunosuppressive marker PD-L1. Stat1-deficient mice displayed increased oral lesion incidence and multiplicity during tumour carcinogenesis in vivo. Immunosuppressive markers PD-1 in CD8+ T cells and PD-L1 in monocytic MDSCs and macrophages were reduced in oral tumours and draining lymph nodes of tumour-bearing Stat1-deficient mice. However, STAT1 was required for anti-tumour functions of T cells during HNSCC in vivo. Finally, we identified TRIM24 to be a negative regulator of STAT1 that plays a similar tumorigenic function to STAT1 in vitro and thus may be a potential target when treating HNSCC. CONCLUSION: Our findings indicate that STAT1 activity plays an important role in tumorigenicity and immunosuppression during HNSCC development.

Ginger extract activates caspase independent paraptosis in cancer cells via ER stress, mitochondrial dysfunction, AIF translocation and DNA damage.

The rhizome of ginger (Zingiber officinale) a common culinary agent is also known for its medicinal activity. We have earlier reported that pure 6-shogaol, an important component of ginger induces paraptosis in triple negative breast cancer (MDA-MB-231) and non small cell lung (A549) cancer cells. However, the chemopreventive potential of the whole ginger extract in food remains to be elucidated. Here, we demonstrate for the first time that ginger extract (GE) triggers similar anticancer activity/paraptosis against the same cell lines but through different molecular mechanisms. Q-TOF LC-MS analysis of the extract showed the presence of several other metabolites along with 6-shogaol and 6-gingerol. GE induces cytoplasmic vacuolation through ER stress and dilation of the ER. Drastic decrease in the mitochondrial membrane potential and ATP production along with the excess generation of ROS contributed to mitochondrial dysfunction. Consequently, GE caused the translocation of apoptosis inducing factor to the nucleus leading to the fragmentation of DNA. Taken together, these show a novel mechanism for ginger extract induced cancer cell death that can be of potential interest for cancer preventive strategies.

Proteasomal dysfunction and ER stress triggers 2'-hydroxy-retrochalcone-induced paraptosis in cancer cells.

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2'-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.

Identification of carbonylated proteins from monocytic cells under diabetes-induced stress conditions.

Diabetes is a metabolic disorder characterized by the presence of elevated glucose in the blood and enhanced oxidative stress. It affects the cellular homeostasis that leads to the development of micro-and macro-vascular complications. Monocytes are the primary immune cells present in the circulatory system. Under high-glucose conditions, the cells undergo oxidative stress and secrete reactive oxygen species. The enhanced release of reactive species is known to modify biomolecules like proteins and nucleic acids. Protein carbonylation, one of the most harmful and irreversible protein modifications, is considered as a key player in the progression of diabetes and associated complications. Hence, the present study explores the identification of carbonylated proteins from the monocytes under diabetic stress and determination of their site of modification. Combined avidin affinity chromatography and bottom-up proteomics experiments identified 13 consistently expressed carbonylated proteins. Most of the identified proteins were reported to have altered functions under diabetic conditions that contribute to the development of diabetes-associated inflammation and complications. We were able to determine oxidative stress-induced modifications on Lys, Val, Ile, Cys, Thr and Asp residues.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

An α, ß-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of ß5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells.

The Association of Adiposity and RAAS with Incident Diabetes in African Americans: The Jackson Heart Study.

BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) and adiposity measures are independently associated with the development of diabetes in African American (AA) adults. However, studies have not examined the combined interaction between RAAS and adiposity measures in relation to diabetes risk in AA adults. OBJECTIVE: We examined the longitudinal association of combined RAAS and adiposity measures with incident diabetes among AAs in the Jackson Heart Study. METHODS: AA adults were assessed at baseline (2000-2004) and over 12 years of follow-up. RAAS, anthropometric (waist circumference [WC], body mass index [BMI]) and adipokine (adiponectin, leptin, leptin: adiponectin ratio [LAR]) measures were collected at baseline. Aldosterone, WC, and LAR were chosen as the best predictor variables. The final model, adjusting for age, sex, education, occupation, systolic blood pressure, smoking, physical activity and RAAS altering medications, incorporated these variables and their interactions (WC*Aldosterone + LAR*Aldosterone) to explore their impact on incident diabetes. RESULTS: Among 3,220 participants without diabetes at baseline, there were 554 incident cases over a median follow-up of 7.5 years. Aldosterone, WC, and LAR were positively associated with incident diabetes (all p < 0.05). A significant interaction was found between WC and aldosterone with a greater association among individuals with lower WC. This interaction was significant in participants with prediabetes but not in those with normoglycemia. No significant interaction was found between log-LAR and aldosterone with risk of incident diabetes. CONCLUSION: Higher aldosterone in participants is associated with greater risk of diabetes, particularly among individuals with prediabetes and lower WC.

The association of aldosterone and endothelin-1 with incident diabetes among African Americans: The Jackson Heart Study.

Introduction: African Americans (AAs) have the highest prevalence of hypertension among United States racial/ethnic groups. Regulators of blood pressure, such as aldosterone and endothelin-1, impact glucose regulation. The relationship between these factors and incident diabetes is not well elucidated among AAs. Methods: Among 3914 AA participants without prevalent diabetes in the Jackson Heart Study, linear regression models were used to examine cross-sectional associations of exposures (aldosterone, endothelin-1, and a combined aldosterone-endothelin-1 score [2-8]) with glycemic measures (fasting plasma glucose [FPG], HbA1c, homeostatic model assessments of beta cell function [HOMA-ß] and insulin resistance [HOMA-IR]). Longitudinal associations of exposures with incident diabetes were examined using Cox proportional hazard models. Models were adjusted for age, sex, education, occupation, systolic blood pressure, smoking, physical activity, dietary intake, alcohol use and adiponectin. Results: Aldosterone and the combined aldosterone-endothelin score were positively associated with FPG, HOMA-IR, and HOMA-ß (all p < 0.05). Endothelin-1 was negatively associated with FPG but positively associated with HOMA-ß (both p < 0.05). Only the aldosterone-endothelin score was positively associated with HbA1c (p < 0.01). A 1-SD higher serum aldosterone and endothelin-1 was associated with a 22 % and 14 % higher risk of incident diabetes, respectively, while a 1-point higher aldosterone-endothelin score was associated with a 13 % higher risk of incident diabetes after adjustment for diabetes risk factors (all p < 0.01). Conclusions: Aldosterone and endothelin-1, factors integral in blood pressure regulation, may play a significant role in the development of diabetes among AAs.

Effects of Hypocaloric Low-Fat, Ketogenic, and Ketogenic and Ketone Supplement Diets on Aldosterone and Renin.

CONTEXT: Ketogenic diets (KDs) and low-fat diets (LFD) result in similar weight loss, but have differential cardiometabolic effects on lipids and insulin. Generally, weight loss decreases renin-angiotensin-aldosterone system (RAAS) activity. OBJECTIVE: Investigate the effects of KDs with varying sodium content vs LFD on RAAS in overweight and obese adults. METHODS: Twenty-eight participants were randomized 1:1 to a KD + ketone salt supplement (KD + KS) or a KD + placebo (KD + PL) arm with prepared hypocaloric meals. Twelve participants were enrolled in a post hoc LFD arm. Serum renin, aldosterone, and anthropometric and metabolic biomarkers were assessed at 0, 2, 4, and 6 weeks. Linear mixed models with random intercepts were used to compare between group differences controlling for sex and body mass index. RESULTS: Participants had a median age of 33 years, 51% female, weighed 91.3 kg, with body mass index 30.6 kg/m2. At 6 weeks, weight decreased by 6, 8, and 7 kg on average in the KD + KS, KD + PL, and LFD groups, respectively (P < .05). Aldosterone increased by 88% and 144% in the KD + PL and KD + KS groups, respectively, but did not change in the LFD after 6 weeks while renin decreased across groups. Systolic and diastolic blood pressure did not change in the KD + PL and KD + KS groups. Log ketones were positively associated with aldosterone (P < .001). Aldosterone was not associated with cardiovascular measures including blood pressure and ejection fraction (P > .05). CONCLUSION: KD reduced weight and increased aldosterone without worsening cardiometabolic risk factors. Future KD studies are needed to elucidate mechanistic connections between ketones and aldosterone.

Adiposity, aldosterone and plasma renin activity among African Americans: The Jackson Heart Study.

Objective: To analyze associations between adiposity and the renin-angiotensin-aldosterone system (RAAS) in a large African American (AA) cohort. Methods: Cross-sectional associations of adiposity (body mass index [BMI], waist circumference [WC], waist:height ratio, waist:hip ratio, leptin, adiponectin, leptin:adiponectin ratio [LAR], subcutaneous [SAT] and visceral adipose tissue [VAT], and liver attenuation [LA]) with aldosterone, plasma renin activity (renin), and aldosterone:renin ratio (ARR) were assessed in the Jackson Heart Study using adjusted linear regression models. Results: A 1-SD higher BMI was associated with a 4.8 % higher aldosterone, 9.4 % higher renin, and 5.0 % lower ARR (all p < 0.05). Log-leptin had the largest magnitude of association with renin (30.2 % higher) and ARR (9.6 % lower), while the strongest association of aldosterone existed for log-LAR (15.3 % higher) (all 1-SD, p < 0.05). SAT was only associated with renin. VAT was associated with higher aldosterone, renin, and ARR. Liver fat was associated with aldosterone and renin, but not ARR. Associations of WC, BMI, and SAT with aldosterone were greater in men while the association with VAT was greater in women (p-interactions < 0.05). Conclusion: Multiple measures of adiposity are associated with the RAAS in AAs. Further studies should examine the role of RAAS in obesity-driven cardiometabolic diseases.

Plumbagin induces paraptosis in cancer cells by disrupting the sulfhydryl homeostasis and proteasomal function.

Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.

Direct readout protonophore induced selective uncoupling and dysfunction of individual mitochondria within cancer cells.

Concurrently, manipulation of mitochondrial activity and its monitoring have enormous significance in cancer therapy and diagnosis. In this context, a fluorescent probe MitoDP has been developed for validating H2S mediated protonophore (2,4-dinitrophenol, DNP) induced mitochondrial membrane potential change, ROS formation and ATP depletion in cancer cells. The extent of protonophore activation for mitochondrial dysfunction is monitored through fluorescence signalling at 450 nm. The current study provides a proof for the concept of endogenous H2S-mediated controlled and spatial release of bioactive agents, or toxins specifically in mitochondria of cancer cells.

Monitoring of topoisomerase (I) inhibitor camptothecin release from endogenous redox-stimulated GO-polymer hybrid carrier.

We have developed endogenous redox-responsive polymer conjugated GO-based hybrid nanomaterials (GO-PEGssFol-CPT) for delivery of anticancer drug camptothecin (CPT) to the cancer cells. The synthesized intermediate (PEGSSFol) and CPT loaded GO- PEGSSFol were characterized using Fourier transform infrared spectroscopy (FTIR) and 1H NMR. The morphological feature changes of TEM and AFM images have confirmed the loading of CPT on the nanocarrier and its release from the nanocarrier. The amount of CPT was loaded was found to be 14.2%. The extent of camptothecin (CPT) release from GO-BiotinPVA-CPT in the presence of different concentrations of glutathione (GSH) was monitored with the increase in the fluorescence intensity at λmax 438 nm and UV-Vis absorbance at 366 nm. The time-dependent camptothecin (CPT) release was monitored in the presence of GSH. It was noticed that CPT was completely released from GO-PEGssFol-CPT within 45 min. This release process is free from interference by other ubiquitous analytes in the living system. The constant fluorescence intensity of GO-PEGssFol-CPT against acidic pH indicated that CPT would not be released in the extracellular region of cancer cells. Therefore, such delivery system could be used to prevent unwanted cytotoxicity to the healthy cells. The GO-PEGssFol-CPT showed higher antiproliferative activity against cervical cancer cells compared to the CPT. Thus, GO-PEGssFol-CPT can be a new material to deliver the anticancer drug to the target tumor region.

A bioorthogonal fluorescent probe for mitochondrial hydrogen sulfide: new strategy for cancer cell labeling.

We report the application of a chemodosimeter 'turn on' fluorescent probe for detecting endogenous H2S formation in cancer cells. Mito-HS showed a bathochromic shift in the UV-vis-absorption spectrum from 355 nm to 395 nm in the presence of H2S. Furthermore, it showed an ∼43-fold fluorescence enhancement at λem = 450 nm in the presence of H2S (200 µM). The cancer cell-specific fluorescence imaging reveals that Mito-HS has the ability to distinguish cancer cells from normal cells based on the level of endogenous H2S formation. In due course, Mito-HS would be a powerful cancer biomarker based on its ability to estimate endogenous H2S formation in living cells.