Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

Antioxidants and basal cell carcinoma of the skin : a nested case-control study

Objective To investigate the relationship between basal cell carcinoma (BCC) and antioxidant nutrients, specifically carotenoids, vitamin E and selenium.

Methods The Nambour Skin Cancer Study is an ongoing, community-based study of randomly selected adult residents of a township in sub-tropical Queensland, Australia. Using a nested case–control design, incident cases of BCC (n=90) were compared with age and sex matched controls (n=90). Dietary exposure was measured using food frequency questionnaire estimates of intake as well as serum biomarkers. Other determinants of skin cancer including sun exposure were also considered. Dietary intakes were adjusted for energy intake, and serum carotenoids and vitamin E were adjusted for serum cholesterol. Odds ratios were calculated across quartiles of dietary intake and serum biomarkers and linear trends were assessed using logistic regression, adjusting for age, sex and supplement use.

Results and conclusions In this prospective study no significant associations were found between BCC and carotenoids, vitamin E or selenium, as measured by serum biomarkers or dietary intake, although there was a suggestion of a positive association with lutein intake.

Natriuretic peptide regulation of the kidney and urinary bladder in the cane toad, Bufo marinus

The natriuretic peptide system of mammals is important in the control of blood volume but its function in non-mammalian animals is unclear. This study identified a functional natriuretic peptide system in an amphibian and showed that the hormones are involved in the control of fluid balance.

A microfluidic system for testing the responses of head and neck squamous cell carcinoma tissue biopsies to treatment with chemotherapy drugs

Tumors are heterogeneous masses of cells characterized pathologically by their size and spread. Their chaotic biology makes treatment of malignancies hard to generalize. We present a robust and reproducible glass microfluidic system, for the maintenance and “interrogation” of head and neck squamous cell carcinoma (HNSCC) tumor biopsies, which enables continuous media perfusion and waste removal, recreating in vivo laminar flow and diffusion-driven conditions. Primary HNSCC or metastatic lymph samples were subsequently treated with 5-fluorouracil and cisplatin, alone and in combination, and were monitored for viability and apoptotic biomarker release ‘off-chip’ over 7 days. The concentration of lactate dehydrogenase was initially high but rapidly dropped to minimally detectable levels in all tumor samples; conversely, effluent concentration of WST-1 (cell proliferation) increased over 7 days: both factors demonstrating cell viability. Addition of cell lysis reagent resulted in increased cell death and reduction in cell proliferation. An apoptotic biomarker, cytochrome c, was analyzed and all the treated samples showed higher levels than the control, with the combination therapy showing the greatest effect. Hematoxylin- and Eosin-stained sections from the biopsy, before and after maintenance, demonstrated the preservation of tissue architecture. This device offers a novel method of studying the tumor environment, and offers a pre-clinical model for creating personalized treatment regimens.

Epidermal growth factor receptor-tyrosine kinase inhibitors in advanced squamous cell carcinoma of the lung: a meta-analysis.

Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) are well established in treating metastatic pulmonary adenocarcinoma, especially patients with activating EGFR mutations. EGFR mutations are rare in pulmonary squamous cell carcinomas (SCCs). There are conflicting data supporting the efficacy of EGFR-TKIs in advanced lung SCC. We analyzed the impact of EGFR-TKIs on progression-free survival (PFS) and overall survival (OS) in unselected patients with lung SCC.

Genomics based biomarker discovery in oral squamous cell carcinoma

 The present thesis showed signaling mechanisms and pathways essential for oral cancer progression through genomics approach. It has identified markers that are of diagnostic, prognostic and therapeutic importance. It has also shown that aspirin is a potential drug in oral cancer treatment.

Incidence of bladder cancer in Sri Lanka : analysis of the cancer registry data and review of the incidence of bladder cancer in the South Asian population

PurposeTo investigate the incidence of bladder cancer (BC) in Sri Lanka and to compare risk factors and outcomes with those of other South Asian nations and South Asian migrants to the United Kingdom (UK) and the United States (US).Materials and MethodsThe incidence of BC in Sri Lanka was examined by using two separate cancer registry databases over a 5-year period. Smoking rates were compiled by using a population-based survey from 2001 to 2009 and the relative risk was calculated by using published data.ResultsA total of 637 new cases of BC were diagnosed over the 5-year period. Sri Lankan BC incidence increased from 1985 but remained low (1.36 and 0.3 per 100,000 in males and females) and was similar to the incidence in other South Asian countries. The incidence was lower, however, than in migrant populations in the US and the UK. In densely populated districts of Sri Lanka, these rates almost doubled. Urothelial carcinoma accounted for 72%. The prevalence of male smokers in Sri Lanka was 39%, whereas Pakistan had higher smoking rates with a 6-fold increase in BC.ConclusionsSri Lankan BC incidence was low, similar to other South Asian countries (apart from Pakistan), but the actual incidence is likely higher than the cancer registry rates. Smoking is likely to be the main risk factor for BC. Possible under-reporting in rural areas could account for the low rates of BC in Sri Lanka. Any genetic or environmental protective effects of BC in South Asians seem to be lost on migration to the UK or the US and with higher levels of smoking, as seen in Pakistan.

Functional analysis of natriuretic peptide receptors in the bladder of the toad, bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific ¹²⁵I-rat atrial natriuretic peptide (¹²⁵I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with ¹²⁵I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to ¹²⁵I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Studies of the human lymphocyte P2Z receptor and its activation of phospholipase D

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.

Balloon cell melanoma: a case report with polarized and non-polarized dermatoscopy and dermatopathology

Balloon cell melanoma is a rare melanoma subtype, with only one previous case with dermatoscopy published. It is often non-pigmented, leading to diagnostic difficulty, and there is a tendency for lesions to be thick at diagnosis. We report a case of balloon cell melanoma on the forearm of a 61-year-old man with both polarized and non-polarized dermatoscopy and dermatopathology. It presented as a firm pale nodule with focal eccentric pigmentation. The clinical images evoke a differential diagnosis of dermatofibroma, dermal nevus, Spitz nevus and basal cell carcinoma as well as melanoma. This melanoma was partially pigmented due to a small, pigmented superficial spreading component on the edge of the non-pigmented balloon cell nodule, prompting further evaluation. In retrospect there was the clue to malignancy of polarizing-specific white lines (chrysalis structures) and polymorphous vessels, including a pattern of dot vessels. The reticular lines exclude basal cell carcinoma, polarizing-specific white lines are inconsistent with the diagnosis of dermal nevus and their eccentric location is inconsistent with both Spitz nevus and dermatofibroma. Excision biopsy was performed, revealing a superficial spreading melanoma with two distinct invasive components, one of atypical non-mature epithelioid cells and the other an amelanotic nodular component, comprising more than 50% of the lesion, characterized by markedly distended epithelioid melanocytes showing pseudo-xanthomatous cytoplasmic balloon cell morphology. A diagnosis of balloon cell melanoma, Breslow thickness 1.9 mm, mitotic rate 3 per square millimeter was rendered. Wide local excision was performed, as was sentinel lymph node biopsy, which was negative.

Increasing BMI was associated with increasing risk for overall cancer incidence and mortality in middle-aged women

Question: In middle-aged women, what is the association between body mass index (BMI) and cancer incidence and mortality?METHODSDesign: Prospective cohort study with mean follow-up of 5.4 years for cancer incidence and 7.0 years for cancer mortality.Setting: Population-based study in England and Scotland, United Kingdom.Patients: 1 222 630 women 55 to 64 years of age (mean 56 y) who had no history of cancer at baseline.Risk factors: BMI at baseline, divided into 5 categories (< 22.5, 22.5 to 24.9, 25.0 to 27.4, 27.5 to 29.9, and ≥ 30 kg/m2).Outcomes: Cancer incidence and mortality, overall and for 17 specific types of cancer, identified through linkage with the National Health Service central registers.Main results: Increasing BMI was associated with increasing risks for all cancers, endometrial cancer, esophageal adenocarcinoma, kidney cancer, leukemia, postmenopausal breast cancer, multiple myeloma, pancreatic cancer, non-Hodgkin lymphoma, and ovarian cancer; and with decreasing risks for esophageal squamous cell carcinoma, lung cancer, and premenopausal breast cancer (Table). Risks for stomach, colorectal, cervical, bladder, and brain cancer and malignant melanoma did not vary by BMI. Patterns for cancer mortality were similar to those for cancer incidence: Relative risk for death from any type of cancer was 1.06 (95% CI 1.02 to 1.10) per 10-unit increase in BMI. In postmenopausal women, the estimated proportion of cancer attributable to being overweight or obese (BMI ≥ 25 kg/m2) was 5% for all cancers and about 50% for endometrial cancer and esophageal adenocarcinoma.Conclusions: In middle-aged women, increasing body mass index was associated with increasing risk for cancer incidence and mortality overall. High body mass index increased risk for some types of cancer but reduced risk for other types.

Development of microfluidic-based analytical methodology for studying the effects of chemotherapy agents on cancer tissue

Whilst a multitude of techniques have been employed to study the biology of tumour tissue and its response to chemotherapeutic reagents, most current methodologies do not capture the sophistication of the in vivo environment. Microfluidics however offers the ability to maintain and interrogate primary tissue samples in an environment with biomimetic flow characteristics. In this study head and neck squamous cell carcinoma (HNSCC) tumour biopsies have been used to investigate the performance of a microfluidic device for generating clinically-useful information. The response of fresh and cryogenically-frozen primary HNSCC or metastatic lymph node samples to chemotherapy drugs (cisplatin, 5-flurouracil or docetaxel), alone and in combination, were monitored for both proliferation (water-soluble tetrazolium salt metabolism) and cell death biomarker release (lactate dehydrogenase, LDH) “off-chip”. The frozen tissue showed no significant difference in terms of either proliferation or LDH release in comparison with the matched fresh samples. Administration of all drugs caused cell death, in a dose-response manner, with the combination showing the greatest amount of cytotoxicity particularly at days 8 and 9; correlating well with published clinical data. The system described here offers an innovative method for studying the tumour microenvironment in vitro and, through incorporation of relevant analytical modules, provides the basis of a pre-clinical device that can be used to define personalised treatment regimens.

Bardoxolone methyl induces apoptosis and autophagy and inhibits epithelial-to-mesenchymal transition and stemness in esophageal squamous cancer cells

Natural and synthetic triterpenoids have been shown to kill cancer cells via multiple mechanisms. The therapeutic effect and underlying mechanism of the synthetic triterpenoid bardoxolone methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me) on esophageal cancer are unclear. Herein, we aimed to investigate the anticancer effects and underlying mechanisms of CDDO-Me in human esophageal squamous cell carcinoma (ESCC) cells. Our study showed that CDDO-Me suppressed the proliferation and arrested cells in G2/M phase, and induced apoptosis in human ESCC Ec109 and KYSE70 cells. The G2/M arrest was accompanied with upregulated p21Waf1/Cip1 and p53 expression. CDDO-Me significantly decreased B-cell lymphoma-extra large (Bcl-xl), B-cell lymphoma 2 (Bcl-2), cleaved caspase-9, and cleaved poly ADP ribose polymerase (PARP) levels but increased the expression level of Bcl-2-associated X (Bax). Furthermore, CDDO-Me induced autophagy in both Ec109 and KYSE70 cells via suppression of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. There were interactions between the autophagic and apoptotic pathways in Ec109 and KYSE70 cells subject to CDDO-Me treatment. CDDO-Me also scavenged reactive oxygen species through activation of the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) pathway in Ec109 and KYSE70 cells. CDDO-Me inhibited cell invasion, epithelial-mesenchymal transition, and stemness in Ec109 and KYSE70 cells. CDDO-Me significantly downregulated E-cadherin but upregulated Snail, Slug, and zinc finger E-box-binding homeobox 1 (TCF-8/ZEB1) in Ec109 and KYSE70 cells. CDDO-Me significantly decreased the expression of octamer-4, sex determining region Y-box 2 (Sox-2), Nanog, and B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), all markers of cancer cell stemness, in Ec109 and KYSE70 cells. Taken together, these results indicate that CDDO-Me is a promising anticancer agent against ESCC. Further studies are warranted to explore the molecular targets, efficacy and safety of CDDO-Me in the treatment of ESCC.