An Experimental Protocol for the Boyden Chamber Invasion Assay With Absorbance Readout.

The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology. Key features • Measurement of tumor invasion using human cancer cells. • Ability to measure the pro-invasive/anti-invasive activity of small molecules and biological modifiers. • Measurement of chemotaxis, chemokines, trafficking of immune cells, and proteolytic activity of matrix metalloproteinases, lysosomal hydrolysates, collagenases, and plasminogen activators in physiological and pathological conditions. • Investigation of the role of extracellular matrix proteins in the crosstalk between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma.

The Flavoring Agent Ethyl Vanillin Induces Cellular Stress Responses in HK-2 Cells.

Flavored e-cigarettes are a popular alternative to cigarette smoking; unfortunately, the extrapulmonary effects are not well-characterized. Human proximal tubule cells were cultured for 24 or 48 h with 0-1000 µM ethyl vanillin (ETH VAN) and cytotoxicity evaluated. Mitochondrial health was significantly diminished following 48 h of exposure, accompanied by significantly decreased spare capacity, coupling efficiency, and ATP synthase expression. ETH VAN at 24 h inhibited glycolysis. The endoplasmic reticulum (ER) stress marker C/EBP homologous protein (CHOP) was increased at 100 µM relative to 500-1000 µM. The downstream proapoptotic marker cleaved caspase-3 subsequently showed a decreasing trend in expression after 48 h of exposure. The autophagy biomarkers microtubule-associated proteins 1A/1B light chain 3 (LC3B-I and LC3B-II) were measured by Western blot. LC3B-II levels and the LC3B-II/LC3B-I ratio increased at 24 h, which suggested activation of autophagy. In contrast, by 48 h, the autophagy biomarker LC3B-II decreased, resulting in no change in the LC3B-II/LC3B-I ratio. Mitophagy biomarker PTEN-induced putative kinase 1 (PINK1) expression decreased after 48 h of exposure. The downstream marker Parkin was not significantly changed after 24 or 48 h. These findings indicate that the flavoring ETH VAN can induce energy pathway dysfunction and cellular stress responses in a renal model.

The e-liquid flavoring cinnamaldehyde induces cellular stress responses in human proximal tubule (HK-2) kidney cells.

Flavored e-liquid use has become popular among e-cigarette users recently, but the effects of such products outside the lung are not well characterized. In this work, acute exposure to the popular flavoring cinnamaldehyde (CIN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0-100 µM CIN for 24-48 h and cellular stress responses were assessed. Mitochondrial viability via MTT assay was significantly decreased at 20 µM for 24 and 48 h exposure. Seahorse XFp analysis showed significantly decreased mitochondrial energy output at 20 µM by 24 h exposure, in addition to significantly reduced ATP Synthase expression. Seahorse analysis also revealed significantly decreased glycolytic function at 20 µM by 24 h exposure, suggesting inability of glycolytic processes to compensate for reduced mitochondrial energy output. Cleaved caspase-3 expression, a mediator of apoptosis, was significantly increased at the 24 h mark. C/EBP homologous protein (CHOP) expression, a mediator of ER-induced apoptosis, was induced by 48 h and subsequently lost at the highest concentration of 100 µM. This decrease was accompanied by a simultaneous decrease in its downstream target cleaved caspase-3 at the 48 h mark. The autophagy marker microtubule-associated protein 1 A/1B light chain 3 (LC3B-I and LC3B-II) expression was significantly increased at 100 µM by 24 h. Autophagy-related 7 (ATG7) protein and mitophagy-related proteins PTEN-induced putative kinase 1 (PINK1) and PARKIN expression were significantly reduced at 24 and 48 h exposure. These results indicate acute exposure to CIN in the kidney HK-2 model induces mitochondrial dysfunction and cellular stress responses.

The E-liquid flavoring vanillin alters energy and autophagic pathways in human proximal tubule (HK-2) epithelial cells.

The use of flavored e-liquids in electronic nicotine delivery systems (ENDS) has become very popular in recent years, but effects of these products have not been well characterized outside the lung. In this study, acute exposure to the popular flavoring vanillin (VAN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0-1000 µM VAN for 24 or 48 h and cellular stress responses were determined. Mitochondrial viability using MTT assay showed a significant decrease between the control and 1000 µM group by 48 h. Seahorse XFp analysis showed significantly increased basal respiration, ATP production, and proton leak after 24 h exposure. By 48 h exposure, these parameters remained significantly increased in addition to non-mitochondrial respiration and maximal respiration. Glycolytic activity after 24 h exposure showed significant decreases in glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. The autophagy markers microtubule-associated protein 1A/1B light chain 3 (LC3B-I and LC3B-II) were probed via western blotting. The ratio of LC3B-II/LC3B-I was significantly increased after 24 h exposure to VAN, but by 48 h this ratio significantly decreased. The mitophagy marker PINK1 showed an increasing trend at 24 h, and its downstream target Parkin was significantly increased between the control and 750 µM group only. Finally, the oxidative stress marker 4-HNE was significantly decreased after 48 h exposure to VAN. These results indicate that acute exposure to VAN in the kidney HK-2 model can induce energy and autophagic changes within the cell.

mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

Anticancer Activity of Region B Capsaicin Analogs.

The heterocyclic vanilloid compound capsaicin is responsible for the spicy and pungent flavor of chili peppers. Several convergent studies have shown that capsaicin suppresses the growth of multiple human cancers. Apart from capsaicin, natural and synthetic capsaicin-like compounds display growth suppressive activity in human cancers. The pharmacophore of capsaicin is comprised of three regions, namely region A (the aromatic ring), region B (the amide bond), and region C (the side chain). The present manuscript describes the isolation and synthesis of capsaicin analogs which have structural modifications in region B of the molecule. Furthermore, the pharmacokinetic properties, anticancer activity of region B capsaicin analogs, as well as the signaling pathways (underlying the growth-inhibitory effects of region B capsaicin analogs) have also been described. The discovery of novel, second-generation region B capsaicin analogs may foster the hope of innovative nutrition-based combination therapies in human cancers.

Spherical Invasion Assay: A Novel Method to Measure Invasion of Cancer Cells.

The invasion of tumor cells into the neighboring blood vessels and lymph nodes is a vital step for distant metastasis. Traditionally, the invasive activity of growth factors (or the anti-invasive activity of drugs) is measured with the Boyden chamber assay. However, this assay has a few disadvantages like poor physiological relevance of transwell inserts and an inability to control chemokine gradients. The Boyden chamber assay is one of the most prevalent methods to measure the invasion of cancer cells. It would be advantageous to develop another assay that could validate the results of the Boyden chamber assay. With this in mind, our laboratory developed the spherical invasion assay (SIA) to measure the pro-invasive activity of human cancer cells. The SIA also circumvents some of the drawbacks of the Boyden chamber assay. The present manuscript measures the anti-invasive activity of the Src kinase inhibitor PP2 in A549 human non-small cell lung carcinoma (NSCLC) cells using the SIA. The SIA protocol is comprised of two steps. In the first step, A549 human NSCLC cells (treated or not with PP2) were mixed with Matrigel and seeded in the middle of an eight-well chamber slide. After 24 h, a second layer of Matrigel was overlaid over the first layer. Over the course of the next 24 h, the A549 cells invade from the primary to the secondary Matrigel layers. Subsequently, the cells are visualized by phase-contrast microscopy and the images obtained are quantified using ImageJ to calculate the anti-invasive activity of PP2 in A549 cells. The results of the SIA correlate well with Boyden chamber assays. The SIA may be adapted for multiple experimental designs, such as drug screening (to combat invasion and metastasis), measuring the pro-invasive activity of growth factors, and elucidating the signaling pathways underlying the pro-invasive/anti-invasive activity of biological modifiers. Graphic abstract: Diagrammatic illustration of the spherical invasion assay ( Hurley et al., 2017 ) . A. The first layer is comprised of human cancer cells mixed in a 1:1 suspension with Phenol Red containing Matrigel (represented as LAYER 1 in the figure). After 24 h, the cancer cells grow and extend up to the boundary of this first layer. B. A second layer of 1:1 solution Phenol Red-free Matrigel, in Phenol Red-free RPMI (represented as LAYER 2 in the figure) is added on top of the first Matrigel spot. The cells are incubated for 24 h at 37°C. C. Over these 24 h, the cancer cells invade from the primary layer into the secondary Matrigel layer. The chamber slides are observed by phase-contrast microscopy. D. A representative photograph of the images obtained by the SIA is shown. The black arrow indicates the cancer cells invading into the second layer of Matrigel. The dotted line represents the interface between the two layers. The distance to which the cells have traveled (into the secondary Matrigel layer) is measured at ten sites (for each photograph) in a randomized double-blind fashion by three independent observers, using NIH ImageJ Version 1.47. This process is repeated for three separate photographic fields per sample.

Anti-cancer activity of sustained release capsaicin formulations.

Capsaicin (trans-8-methyl-N-vanillyl-6-noneamide) is a hydrophobic, lipophilic vanilloid phytochemical abundantly found in chili peppers and pepper extracts. Several convergent studies show that capsaicin displays robust cancer activity, suppressing the growth, angiogenesis and metastasis of several human cancers. Despite its potent cancer-suppressing activity, the clinical applications of capsaicin as a viable anti-cancer drug have remained problematic due to its poor bioavailability and aqueous solubility properties. In addition, the administration of capsaicin is associated with adverse side effects like gastrointestinal cramps, stomach pain, nausea and diarrhea and vomiting. All these hurdles may be circumvented by encapsulation of capsaicin in sustained release drug delivery systems. Most of the capsaicin-based the sustained release drugs have been tested for their pain-relieving activity. Only a few of these formulations have been investigated as anti-cancer agents. The present review describes the physicochemical properties, bioavailability, and anti-cancer activity of capsaicin-sustained release agents. The asset of such continuous release capsaicin formulations is that they display better solubility, stability, bioavailability, and growth-suppressive activity than the free drug. The encapsulation of capsaicin in sustained release carriers minimizes the adverse side effects of capsaicin. In summary, these capsaicin-based sustained release drug delivery systems have the potential to function as novel chemotherapies, unique diagnostic imaging probes and innovative chemosensitization agents in human cancers.

Nonpungent N-AVAM Capsaicin Analogues and Cancer Therapy.

Capsaicin displays robust growth-inhibitory activity in multiple human cancers. However, the feasibility of capsaicin as a clinically relevant anticancer drug is hampered by its adverse side effects. This concern has led to extensive research focused on the isolation and synthesis of second-generation nonpungent capsaicin analogues with potent antineoplastic activity. A major class of nonpungent capsaicin-like compounds belongs to the N-acyl-vanillylamide (N-AVAM) derivatives of capsaicin (hereafter referred as N-AVAM capsaicin analogues). This perspective discusses the isolation of N-AVAM capsaicin analogues from natural sources as well as their synthesis by chemical and enzymatic methods. The perspective describes the pharmacokinetic properties and anticancer activity of N-AVAM capsaicin analogues. The signaling pathways underlying the growth-inhibitory effects of N-AVAM capsaicin analogues have also been highlighted. It is hoped that the insights obtained in this perspective will facilitate the synthesis of a second generation of N-AVAM capsaicin analogues with improved stability and growth-suppressive activity in human cancer.

Capsaicinoids: Multiple effects on angiogenesis, invasion and metastasis in human cancers.

Cancer progression is a complex multistep process comprising of angiogenesis of the primary tumor, its invasion into the surrounding stroma and its migration to distant organs to produce metastases. Nutritional compounds of the "capsaicinoid" family regulate angiogenesis, invasion and metastasis of tumors. Capsaicinoids display robust anti-angiogenic activity in both cell culture and mice models. However, conflicting reports exist about the effect of capsaicinoids on invasion of metastasis of cancers. While some published reports have described an anti-invasive and anti-metastatic role for capsaicinoids, others have argued that capsaicinoids stimulate invasion and metastasis of cancers. The present review article summarizes these findings involving the bioactivity of capsaicin in angiogenesis, invasion and metastasis of cancer. A survey of literature indicate that they are several articles summarizing the growth-inhibitory activity of capsaicinoids but few describe its effects on angiogenesis, invasion and metastasis in detail. Our review article fills this gap of knowledge. The discovery of a second generation of natural and synthetic capsaicin analogs (with anti-tumor activity) will pave the way to improved strategies for the treatment of several human cancers.

Capsaicinoids enhance chemosensitivity to chemotherapeutic drugs.

Cytotoxic chemotherapy is the mainstay of cancer treatment. Conventional chemotherapeutic agents do not distinguish between normal and neoplastic cells. This leads to severe toxic side effects, which may necessitate the discontinuation of treatment in some patients. Recent research has identified key molecular events in the initiation and progression of cancer, promoting the design of targeted therapies to selectively kill tumor cells while sparing normal cells. Although, the side effects of such drugs are typically milder than conventional chemotherapies, some off-target effects still occur. Another serious challenge with all chemotherapies is the acquisition of chemoresistance upon prolonged exposure to the drug. Therefore, identifying supplementary agents that sensitize tumor cells to chemotherapy-induced apoptosis and help minimize drug resistance would be valuable for improving patient tolerance and response to chemotherapy. The use of effective supplementary agents provides a twofold advantage in combination with standard chemotherapy. First, by augmenting the activity of the chemotherapeutic drug it can lower the dose needed to kill tumor cells and decrease the incidence and severity of treatment-limiting side effects. Second, adjuvant therapies that lower the effective dose of chemotherapy may delay/prevent the development of chemoresistance in tumors. Capsaicinoids, a major class of phytochemical compounds isolated from chili peppers, have been shown to improve the efficacy of several anti-cancer drugs in cell culture and animal models. The present chapter summarizes the current knowledge about the chemosensitizing activity of capsaicinoids with conventional and targeted chemotherapeutic drugs, highlighting the potential use of capsaicinoids in novel combination therapies to improve the therapeutic indices of conventional and targeted chemotherapeutic drugs in human cancers.

Acetylcholine signaling system in progression of lung cancers.

The neurotransmitter acetylcholine (ACh) acts as an autocrine growth factor for human lung cancer. Several lines of evidence show that lung cancer cells express all of the proteins required for the uptake of choline (choline transporter 1, choline transporter-like proteins) synthesis of ACh (choline acetyltransferase, carnitine acetyltransferase), transport of ACh (vesicular acetylcholine transport, OCTs, OCTNs) and degradation of ACh (acetylcholinesterase, butyrylcholinesterase). The released ACh binds back to nicotinic (nAChRs) and muscarinic receptors on lung cancer cells to accelerate their proliferation, migration and invasion. Out of all components of the cholinergic pathway, the nAChR-signaling has been studied the most intensely. The reason for this trend is due to genome-wide data studies showing that nicotinic receptor subtypes are involved in lung cancer risk, the relationship between cigarette smoke and lung cancer risk as well as the rising popularity of electronic cigarettes considered by many as a "safe" alternative to smoking. There are a small number of articles which review the contribution of the other cholinergic proteins in the pathophysiology of lung cancer. The primary objective of this review article is to discuss the function of the acetylcholine-signaling proteins in the progression of lung cancer. The investigation of the role of cholinergic network in lung cancer will pave the way to novel molecular targets and drugs in this lethal malignancy.

Anticancer Activity of Natural and Synthetic Capsaicin Analogs.

The nutritional compound capsaicin is the major spicy ingredient of chili peppers. Although traditionally associated with analgesic activity, recent studies have shown that capsaicin has profound antineoplastic effects in several types of human cancers. However, the applications of capsaicin as a clinically viable drug are limited by its unpleasant side effects, such as gastric irritation, stomach cramps, and burning sensation. This has led to extensive research focused on the identification and rational design of second-generation capsaicin analogs, which possess greater bioactivity than capsaicin. A majority of these natural capsaicinoids and synthetic capsaicin analogs have been studied for their pain-relieving activity. Only a few of these capsaicin analogs have been investigated for their anticancer activity in cell culture and animal models. The present review summarizes the current knowledge of the growth-inhibitory activity of natural capsaicinoids and synthetic capsaicin analogs. Future studies that examine the anticancer activity of a greater number of capsaicin analogs represent novel strategies in the treatment of human cancers.

Capsaicin synergizes with camptothecin to induce increased apoptosis in human small cell lung cancers via the calpain pathway.

Small cell lung cancer (SCLC) is characterized by excellent initial response to chemotherapy and radiation therapy with a majority of the patients showing tumor shrinkage and even remission. However, the challenge with SCLC therapy is that patients inevitably relapse and subsequently do not respond to the first line treatment. Recent clinical studies have investigated the possibility of camptothecin-based combination therapy as first line treatment for SCLC patients. Conventionally, camptothecin is used for recurrent SCLC and has poor survival outcomes. Therefore, drugs which can improve the therapeutic index of camptothecin should be valuable for SCLC therapy. Extensive evidence shows that nutritional compounds like capsaicin (the spicy compound of chili peppers) can improve the anti-cancer activity of chemotherapeutic drugs in both cell lines and animal models. Statistical analysis shows that capsaicin synergizes with camptothecin to enhance apoptosis of human SCLC cells. The synergistic activity of camptothecin and capsaicin is observed in both classical and variant SCLC cell lines and, in vivo, in human SCLC tumors xenotransplanted on chicken chorioallantoic membrane (CAM) models. The synergistic activity of capsaicin and camptothecin are mediated by elevation of intracellular calcium and the calpain pathway. Our data foster hope for novel nutrition based combination therapies in SCLC.

Non-pungent long chain capsaicin-analogs arvanil and olvanil display better anti-invasive activity than capsaicin in human small cell lung cancers.

The nutritional compound capsaicin inhibits the invasion of many types of human cancers. The clinical development of capsaicin as an anti-cancer drug is limited due to its unfavorable side effects like burning sensation, stomach cramps, gut pain and nausea. This study compared the anti-invasive activity of capsaicin to non-pungent long chain capsaicin analogs, namely arvanil and olvanil, in human small cell lung cancer cells. Boyden chamber invasion assays revealed that arvanil and olvanil displayed improved anti-invasive activity relative to capsaicin in human SCLC cells. The results of the Boyden chamber assay were confirmed by the spherical invasion assay, and similar results were obtained. The anti-invasive activity of arvanil, olvanil and capsaicin were independent of TRPV and CB1 receptors. Furthermore, the anti-invasive activity of arvanil, olvanil and capsaicin was mediated by the AMPK pathway. Depletion of AMPK levels by siRNA methodology abrogated the anti-invasive activity of arvanil, olvanil and capsaicin. The non-pungent capsaicin analogs arvanil and olvanil display improved anti-invasive activity relative to capsaicin in human SCLC cells. These agents may represent the second generation of capsaicin-like compounds which are more potent than the parent molecule and have a better side effect profile.

The flavonoid nobiletin inhibits tumor growth and angiogenesis of ovarian cancers via the Akt pathway.

Despite its importance, the death rate of ovarian cancer has remained unchanged over the past five decades, demanding an improvement in prevention and treatment of this malignancy. With no known carcinogens, targeted prevention is currently unavailable, and efforts in early detection of this malignancy by screening biomarkers have failed. The inhibition of angiogenesis, also known as angioprevention, is a promising strategy to limit the growth of solid tumors, including ovarian cancers. Nobiletin, a polymethoxy flavonoid compound isolated from the tiansheng plant, has been shown to inhibit the growth of multiple types of human cancers. However, there are no reports involving the effect on nobiletin on human ovarian cancer. The present report shows that nobiletin potently decreases the viability of ovarian cancer cells in vitro. However, nobiletin does not affect the viability of normal ovarian epithelial cells at <40 µM. The antitumor activity of nobiletin was also observed in athymic mouse models and in chicken chorioallantoic membrane (CAM) models. The anti-neoplastic activity of nobiletin was due to its ability to inhibit angiogenesis. We also studied the molecular mechanisms by which nobiletin suppresses angiogenesis. We observed that nobiletin inhibits secretion of the key angiogenesis mediators, Akt, HIF-1α, NF-κB and vascular epithelial growth factor (VEGF) by ovarian cancer cells. Transient transfection experiments showed that nobiletin inhibits production of HIF-1α by downregulation of Akt. Such decreased levels of HIF-1α were responsible for nobiletin-induced suppression of VEGF. Our data suggest that nobiletin may be a promising anti-angiogenic agent relevant for therapy of ovarian cancers.

Bioavailability of capsaicin and its implications for drug delivery.

The dietary compound capsaicin is responsible for the "hot and spicy" taste of chili peppers and pepper extracts. It is a valuable pharmacological agent with several therapeutic applications in controlling pain and inflammation. Emerging studies show that it displays potent anti-tumor activity in several human cancers. On a more basic research level, capsaicin has been used as a ligand to activate several types of ion-channel receptors. The pharmacological activity of capsaicin-like compounds is dependent on several factors like the dose, the route of administration and most importantly on its concentration at target tissues. The present review describes the current knowledge involving the metabolism and bioavailability of capsaicinoids in rodents and humans. Novel drug delivery strategies used to improve the bioavailability and therapeutic index of capsaicin are discussed in detail. The generation of novel capsaicin-mimetics and improved drug delivery methods will foster the hope of innovative applications of capsaicin in human disease.

Capsaicin induces apoptosis in human small cell lung cancer via the TRPV6 receptor and the calpain pathway.

Capsaicin, the pungent ingredient of chili peppers, displays potent anti-neoplastic activity in a wide array of human cancer cells. The present manuscript examines the signaling pathways underlying the apoptotic activity of capsaicin in human small cell lung cancer (SCLC) in vitro and in vivo. Studies in neuronal cells show that capsaicin exerts its biological activity via the transient receptor potential vanilloid (TRPV) superfamily of cation-channel receptors. The TRPV family is comprised of six members (TRPV1-6). Capsaicin is a known agonist of the TRPV1 receptor. We observed that capsaicin-induced apoptosis in human SCLC cells was mediated via the TRPV receptor family; however it was independent of TRPV1. Surprisingly, the apoptotic activity of capsaicin required the TRPV6 receptor. Depletion of TRPV6 receptor by siRNA methodology abolished the apoptotic activity of capsaicin in SCLC cells. Immunostaining and ELISA showed that TRPV6 receptor was robustly expressed on human SCLC tissues (from patients) and SCLC cell lines but almost absent in normal lung tissues. This correlates with our results that capsaicin induced very little apoptosis in normal lung epithelial cells. The pro-apoptotic activity of capsaicin was mediated by the intracellular calcium and calpain pathway. The treatment of human SCLC cells with capsaicin increased the activity of calpain 1 and 2 by threefold relative to untreated SCLC cells. Such calpain activation, in response to capsaicin, was downstream of the TRPV6 receptor. Taken together, our data provide insights into the mechanism underlying the apoptotic activity of capsaicin in human SCLCs.

Sleep deprivation and error in nurses who work the night shift.

OBJECTIVE: The aim of this study was to investigate the relationship between sleep deprivation and occupational and patient care errors among staff nurses who work the night shift. BACKGROUND: Whereas the aviation and trucking industries report that sleep deprivation increases errors, few studies have examined sleep deprivation association with occupational and patient care errors among nurses. METHODS: A cross-sectional correlational design was used to evaluate relationships between sleep deprivation and occupational and patient care errors in 289 hospital night shift nurses. RESULTS: More than half (56%) of the sample reported being sleep deprived. Sleep-deprived nurses made more patient care errors. Testing for associations with occupational errors was not feasible because of the low number of occupational errors reported. CONCLUSION: Interventions to increase the quality and quantity of sleep among hospital night shift nurses are needed. Improved sleep among night shift nurses will reduce the impact of sleep deprivation on patient care errors.

Nicotine induces the up-regulation of the α7-nicotinic receptor (α7-nAChR) in human squamous cell lung cancer cells via the Sp1/GATA protein pathway.

Nicotine, the addictive component of cigarettes, promotes lung cancer proliferation via the α7-nicotinic acetylcholine receptor (α7-nAChR) subtype. The present manuscript explores the effect of nicotine exposure on α7-nAChR levels in squamous cell carcinoma of the lung (SCC-L) in vitro and in vivo. Nicotine (at concentrations present in the plasma of average smokers) increased α7-nAChR levels in human SCC-L cell lines. Nicotine-induced up-regulation of α7-nAChR was confirmed in vivo by chicken chorioallantoic membrane models. We also observed that the levels of α7-nAChR in human SCC-L tumors (isolated from patients who are active smokers) correlated with their smoking history. Nicotine increased the levels of α7-nAChR mRNA and α7-nAChR transcription in human SCC-L cell lines and SCC-L tumors. Nicotine-induced up-regulation of α7-nAChR required GATA4 and GATA6. ChIP assays showed that nicotine induced the binding of GATA4 or GATA6 to Sp1 on the α7-nAChR promoter, thereby inducing its transcription and increasing its levels in human SCC-L. Our data are clinically relevant because SCC-L patients smoked for decades before being diagnosed with cancer. It may be envisaged that continuous exposure to nicotine (in such SCC-L patients) causes up-regulation of α7-nAChRs, which facilitates tumor growth and progression. Our results will also be relevant to many SCC-L patients exposed to nicotine via second-hand smoke, electronic cigarettes, and patches or gums to quit smoking.