Camel Urine Promotes Sensitization to Doxorubicin by Inhibiting Epithelial-Mesenchymal Transition and Modulating NF-κB-Snail Signaling Pathway in Breast Cancer Cells.

BACKGROUND: Camel urine (CU) has been used as traditional treatment in the Arabian Peninsula for centuries. Although, researchers have reported CU anti-cancer effects, the exact mechanism(s) of action involved has not been fully elucidated. The epithelial-mesenchymal transition EMT is a phenotypic switch that promotes the acquisition of a fibroblastoid-like morphology by epithelial tumor cells, resulting in enhanced tumor cell motility and invasiveness. EMT has been shown to contribute to metastasis and chemoresistance of carcinomas. For that, in the present study, we have assessed the potential mechanism (s) by which CU exert its anti-cancer effects and its possible synergistic therapeutic effect with Doxorubicin (DOX) in breast cancer cells. METHODS: Determination of anti-proliferative and apoptosis validation of CU was performed by 3-(4,5-Dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT), annexin-V-fluorescein isothiocyanate assays, and Western blot. EMT protein markers, migration and invasion of cells were determined by Western blot or immunofluorescent staining, Scratch assay, Transwell invasion assay, respectively. RESULTS: CU applied a significant anti-cancer effect on breast cancer cells via induction of DNA damage and apoptosis in a concentration- and time-dependent manner. Also, CU remarkably reversed the EMT by downregulating N-cadherin and Vimentin expression and upregulating E-cadherin expression. As a result, the stemness, migration and invasion of breast cancer cells were also inhibited, which was likely mediated by NF-κB-Snail signalling pathway and its downstream inflammatory effectors. CU successfully enhanced DOX cytotoxicity by reversing EMT which possibly through inhibition of NF-κB-Snail signalling and subsequently inflammation.  Thus, our study provides new mechanistic bases for the therapeutic application of CU that may improve the outcomes of anti-cancer chemotherapy.

The Growth Inhibitory Potential and Antimetastatic Effect of Camel Urine on Breast Cancer Cells In Vitro and In Vivo.

Although it may sound unpleasant, camel urine has been consumed extensively for years in the Middle East as it is believed to be able to treat a wide range of diseases such as fever, cold, or even cancer. People usually take it by mixing small drops with camel milk or take it directly. The project aims to study the effects of camel urine in inhibiting the growth potential and metastatic ability of 4T1 cancer cell line in vitro and in vivo. Based on the MTT result, the cytotoxicity of camel urine against 4T1 cell was established, and it was dose-dependent. Additionally, the antimetastatic potential of camel urine was tested by running several assays such as scratch assay, migration and invasion assay, and mouse aortic ring assay with promising results in the ability of camel urine to inhibit metastatic process of the 4T1 cells. In order to fully establish camel urine's potential, an in vivo study was carried out by treating mice inoculated with 4T1 cells with 2 different doses of camel urine. By the end of the treatment period, the tumor in both treated groups had reduced in size as compared to the control group. Additional assays such as the TUNEL assay, immunophenotyping, cytokine level detection assay, clonogenic assay, and proteome profiler demonstrated the capability of camel urine to reduce and inhibit the metastatic potential of 4T1 cells in vivo. To sum up, further study of anticancer properties of camel urine is justified, as evidenced through the in vitro and in vivo studies carried out. Better results were obtained at higher concentration of camel urine used in vivo. Apart from that, this project has laid out the mechanisms employed by the substance to inhibit the growth and the metastatic process of the 4T1 cell.

APPLICATION OF NONINVASIVE PREGNANCY DIAGNOSIS IN BACTRIAN CAMELS (CAMELUS BACTRIANUS) USING CUBONI REACTION AND BARIUM CHLORIDE TEST.

Pregnancy diagnoses in half-tamed animals are often very complicated. This study aimed to examine the alternative noninvasive and cheap methods of pregnancy diagnosis from urine in domestic Bactrian camels (Camelus bactrianus). Urine from 14 female camels kept in four European zoologic gardens was collected and tested by two chemical tests--Cuboni reaction and barium chloride test. The Cuboni reaction was significantly (P<0.01) affected by the pregnancy status of female camels. The total accuracy of the Cuboni reaction was 70.5% but it increased significantly (P<0.05) in the time leading up to parturition. The accuracy was 100% in the 3rd third of pregnancy. Urine of nonpregnant females did not react with a solution of barium chloride while, contrary to other studies, white precipitates formed mostly (80 to 100%) in urine of pregnant females. This study concluded that the Cuboni reaction is applicable for pregnancy diagnosis in camels.

Survey of the camel urinary proteome by shotgun proteomics using a multiple database search strategy.

We report the first survey of the dromedary camel urinary proteome. Proteins retained from ultrafiltration of urine were analysed by GeLC-MS/MS (SDS-PAGE followed by LC-MS/MS). In the absence of a complete camel genome sequence, the number of protein identifications was maximised by searching three primary sequence databases: Swiss-Prot, alpaca and camel EST. This search strategy enabled the identification of 1274 peptide sequences, of which 735 were found in at least two independent samples. Functional annotations for proteins identified from alpaca and camel EST sequences were mapped from basic local alignment search tool (protein) searches. These 735 peptides, which included many novel sequences found only in the camel EST database, were grouped to 147 protein descriptors. Gene ontology term analysis of human proteins with sequence similarity showed that camel urine may be particularly enriched in proteins from extracellular compartments and vesicles, and with functions that include carbohydrate-binding and peptidase inhibitor activities. If their biological functions are conserved between species, many of the camel urinary proteins could be involved in various stress and immune responses, and some may have antimicrobial activities.

The pharmacokinetics and metabolism of meloxicam in camels after intravenous administration.

The pharmacokinetics and metabolism of meloxicam was studied in camels (Camelus dromedarus) (n = 6) following intravenous (i.v.) administration of a dose of 0.6 mg·kg/body weight. The results obtained (mean ± SD) were as follows: the terminal elimination half-life (t(1/2ß) ) was 40.2 ± 16.8 h and total body clearance (Cl(T) ) was 1.94 ± 0.66 mL·kg/h. The volume of distribution at steady state (V(SS)) was 92.8 ± 13.7 mL/kg. One metabolite of meloxicam was tentatively identified as methylhydroxy meloxicam. Meloxicam and metabolite were excreted unconjugated in urine. Meloxicam could be detected in plasma 10 days following i.v. administration in camels using a sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) method.

Camel urine inhibits the cytochrome P450 1a1 gene expression through an AhR-dependent mechanism in Hepa 1c1c7 cell line.

AIM OF THE STUDY: Drinking camel urine has been used traditionally to treat numerous cases of cancer yet, the exact mechanism was not investigated. Therefore, we examined the ability of three different camel urines (virgin, lactating, and pregnant source) to modulate a well-known cancer-activating enzyme, the cytochrome P450 1a1 (Cyp1a1) in murine hepatoma Hepa 1c1c7 cell line. MATERIALS AND METHODS: The effect of different camel urines, compared to bovine urines, on Cyp1a1 mRNA was determined using real-time polymerase chain reaction. Cyp1a1 protein and catalytic activity levels were determined using Western blot analysis and 7-ethoxyresorufin as a substrate, respectively. The role of aryl hydrocarbon receptor (AhR)-dependent mechanism was determined using electrophoretic mobility shift assay (EMSA) and the AhR-dependent luciferase reporter gene. RESULTS: All types of camel, but not bovine, urines differentially inhibited the induction of Cyp1a1 gene expression by TCDD, the most potent Cyp1a1 inducer and known carcinogenic chemical. Importantly, virgin camel urine showed the highest degree of inhibition at the activity level, followed by lactating and pregnant camel urines. Furthermore, we have shown that virgin camel urine significantly inhibited the TCDD-mediated induction of Cyp1a1 at the mRNA and protein expression levels. Mechanistically, the ability of virgin camel urine to inhibit Cyp1a1 was strongly correlated with its ability to inhibit AhR-dependent luciferase activity and DNA binding as determined by EMSA, suggesting that AhR-dependent mechanism is involved. CONCLUSIONS: The present work provides the first evidence that camel urine but not that of bovine inhibits the TCDD-mediated toxic effect by inhibiting the expression of Cyp1a1, at both transcriptional and post-transcriptional levels through an AhR-dependent mechanism.

The pharmacokinetics, metabolism and urinary detection time of tramadol in camels.

The pharmacokinetics of tramadol in camels (Camelus dromedarius) were studied following a single intravenous (IV) and a single intramuscular (IM) dose of 2.33 mg kg(-1) bodyweight. The drug's metabolism and urinary detection time were also investigated. Following both IV and IM administration, tramadol was extracted from plasma using an automated solid phase extraction method and the concentration measured by gas chromatography-mass spectrometry (GC/MS). The plasma drug concentrations after IV administration were best fitted by an open two-compartment model. However a three-compartment open model best fitted the IM data. The results (means+/-SEM) were as follows: after IV drug administration, the distribution half-life (t(1/2)(alpha)) was 0.22+/-0.05 h, the elimination half-life (t(1/2)(beta)) 1.33+/-0.18 h, the total body clearance (Cl(T)) 1.94+/-0.18 L h kg(-1), the volume of distribution at steady state (Vd(ss)) 2.58+/-0.44 L kg(-1), and the area under the concentration vs. time curve (AUC(0-infinity)) 1.25+/-0.13 mg h L(-1). Following IM administration, the maximal plasma tramadol concentration (C(max)) reached was 0.44+/-0.07 microg mL(-1) at time (T(max)) 0.57+/-0.11h; the absorption half-life (t(1/2 ka)) was 0.17+/-0.03 h, the (t(1/2)(beta)) was 3.24+/-0.55 h, the (AUC(0-infinity)) was 1.27+/-0.12 mg h L(-1), the (Vd(area)) was 8.94+/-1.41 L kg(-1), and the mean systemic bioavailability (F) was 101.62%. Three main tramadol metabolites were detected in urine. These were O-desmethyltramadol, N,O-desmethyltramadol and/or N-bis-desmethyltramadol, and hydroxy-tramadol. O-Desmethyltramadol was found to be the main metabolite. The urinary detection times for tramadol and O-desmethyltramadol were 24 and 48 h, respectively. The pharmacokinetics of tramadol in camels was characterised by a fast clearance, large volume of distribution and brief half-life, which resulted in a short detection time. O-Desmethyltramadol detection in positive cases would increase the reliability of reporting tramadol abuse.

In vivo and in vitro metabolism of dexamethasone in the camel.

The metabolism of dexamethasone (DXM) in the camel was assessed by in vivo and in vitro techniques. Liver samples were collected at the abattoir from camels of either sex, and microsomes were isolated and characterized as to their protein and haemoprotein content as well as for their ability to metabolise several cytochrome P450 model substrates. The expression of different P450 enzymes was evaluated by means of immunoblotting, and the glucuronidating capacity was assessed with 1-naphthol as the substrate. The activity of 11 beta-hydroxysteroid dehydrogenase type 1 was assayed using metyrapone as a model substrate. To examine the in vitro metabolism of DXM, microsomes were incubated with the corticoid in the presence of either a NADPH-generating system or of uridindiphosphoglucuronic acid. In vivo metabolism of DXM was studied in two male camels, injected with a bolus intravenous dose of DXM (0.2 mg/kg body weight) and DXM metabolites were evaluated in urine samples collected at different times after the administration. DXM and metabolites were extracted using solid phase and liquid-liquid extraction, and analysed by liquid chromatography mass spectrometry (LC/MS) and by LC/MS/MS. Comparative results were obtained by in vitro and in vivo studies. Two phase I metabolites were detected: the major one resulted from reduction of the 3-carbonyl group in ring A and the minor metabolite from ring hydroxylation of ring A. Glucuronidation involved both phase I metabolites as well as the parent compound.

The disposition of diclofenac in camels after intravenous administration.

The pharmacokinetics of diclofenac was studied in camels (Camelus dromedarus) (n=6) following intravenous (i.v.) administration of a dose of 2.5 mg kg(-1) body weight. The metabolism and urinary detection time were also studied. The results obtained (median and range) were as follows: the terminal elimination half-life (t(1/2beta)) was 2.35 (1.90-2.73)h, total body clearance (Cl(T)) was 0.17 (0.16-0.21)lh kg(-1). The volume of distribution at steady state (V(SS)) was 0.31 (0.21-0.39)l(-1)kg(-1), the volume of the central compartment of the two compartment pharmacokinetic model (V(C)) was 0.15 (0.11-0.17)l kg(-1). Five metabolites of diclofenac were tentatively identified in urine and were excreted mainly in conjugate form. The main metabolite was identified as hydroxy diclofenac. Both diclofenac and hydroxy diclofenac, appear to be the main elimination route for diclofenac when administered i.v. in camels. Diclofenac could be identified up to 4 days following i.v. administration in camels using a sensitive gas chromatography/mass spectrometry (GC/MS) method.

Comparative pharmacokinetics of diphenhydramine in camels and horses after intravenous administration.

The pharmacokinetics of diphenhydramine (DPHM) was compared in camels (n = 8) and horses (n = 6) following intravenous (i.v.) administration of a dose of 0.625 mg/kg body weight. In addition, the metabolism and urinary detection time of DPHM was evaluated in camels. The data obtained (median and range in brackets) in camels and horses, respectively, were as follows. The terminal elimination half lives (h) were 1.58 (1.13-2.58) and 6.11 (4.80-14.1), and the total body clearances (L/h per kg) were 1.42 (1.13-1.74) and 0.79 (0.66-0.90). The volumes of distribution at steady state (L/kg) were 2.38 (1.58-4.43) and 5.98 (4.60-8.31) and the volumes of the central compartment of the two compartment pharmacokinetic model were 1.58 (0.80-2.54) and 2.48 (1.79-3.17). All the pharmacokinetic parameters in camels were significantly different from those of horses. Five metabolites of DPHM were tentatively identified in the camel's urine. Two metabolites, diphenylmethoxyacetic acid and 1-(4-hydroxyphenyl)-phenylmethoxyacetic acid, were present in the acid fraction. Two metabolites, desamino-DPHM and diphenylmethanol, were identified in the basic fraction, in addition to DPHM itself, which was present mainly as a conjugate. Even after enzymatic hydrolysis, DPHM could be detected for up to 24 h in camels after an i.v. dose of 0.625 mg/kg body weight.

Silica urolithiasis in the dromedary camel in a subtropical climate.

In 1998, two cases of silica urolithiasis occurred in castrated male dromedaries on an intensive camel farm in the Canary Islands. The immediate attributable cause was the ingestion of large amounts of silica in the feed, estimated as 84.44 g/day. An associated cause was the low level of salt in the diet. Daily ingestion of salt from feed and water was estimated to be 21.8 g (8.6 g of sodium). Seventy-six castrated males from the same farm were divided into four groups: group A received 30 g of salt daily; group B received 40 g; group C received 60 g; and group D received no added salt in the diet (control). The animals were maintained on these dietary regimes for 2 years. No animals from groups A, B or C suffered overt urinary retention. One animal from group D had an obstructive urinary retention 10 months after the study commenced. Thus, 52 g of salt daily appears to be sufficient to prevent urinary retention in dromedaries raised in a subtropical climate.

Pharmacokinetics, metabolism and urinary detection time of flunixin after intravenous administration in camels.

The pharmacokinetics of flunixin were determined after an intravenous dose of 1.1 mg/kg body weight in six camels and 2.2 mg/kg body weight in four camels. The data obtained (mean +/- SEM) for the low and high dose, respectively, were as follows: The elimination half-lives (t1/2 beta) were 3.76 +/- 0.24 and 4.08 +/- 0.49 h, the steady state volumes of distribution (Vdss) were 320.61 +/- 38.53 and 348.84 +/- 35.36 mL/kg body weight, total body clearances (ClT) were 88.96 +/- 6.63 and 84.86 +/- 4.95 mL/h/kg body weight and renal clearances (Clr) were 0.52 +/- 0.09 and 0.62 +/- 0.18 mL/h/kg body weight. A hydroxylated metabolite of flunixin was identified by gas chromatography/mass spectrometry (GC/MS) under electron and chemical ionization and its major fragmentation pattern was verified by tandem mass spectrometry (GC/MS/MS) using neutral loss, daughter and parent scan modes. The detection times for flunixin and its hydroxylated metabolite in urine after an intravenous (i.v.) dose of 2.2 mg/kg body weight were 96 and 48 h, respectively.

Regulation of urinary phosphate excretion in camels.

Urinary inorganic phosphorus (P) excretion was measured in 16 adult female non-pregnant, non-lactating daily watered camels. They were randomly divided into four groups of four animals. Groups 1, 2, and received either an i.v. infusion of phosphate or synthetic human parathyroid hormone (PTH) or PTH-related peptide (PTHrP), respectively. The fourth group was used as a control. Intravenous P loading induced a significant increase in phosphatemia and in P renal clearance. Both PTH and PTHrP increased calcemia and decreased phosphatemia. They had no significant effect on urinary calcium excretion, but they increased P renal clearance and phosphaturia. Thus, the regulation of urinary P excretion in normally watered camels looks similar to that already described in other ruminants.

An ultrastructural study of the erythrocytes associated with haematochemical data of the camel Camelus dromedarius.

The aims of the present study were to characterize by electron microscopy the morphological features of the erythrocytes of normal adult and young camels Camelus dromedarius in a renal blood vessel and in a smear. These cells are thin and elliptical, with a slight central elevation. They are slightly larger, but thinner than those reported by other authors. It is found that the renal blood vessels in adults have a thicker basal lamina with the presence of electron dense membranous bodies. These two features were described in different parts of the uriniferous tubules of the camel in previous studies. The numerical data of the haematochemical parameters that are reported here are in general similar for both adult and young camels, while they differ from those of other studies, this may have resulted from their unknown provenance and their previous dietary regime. Moreover, these parameters probably relate to the arid environment of the animal.