The Cytotoxic Effect of Small and Large Molecules of PMF Fraction Extracted from Camel Urine on Cancer Cells

Aim of the work: Animal urine, including that of camels, has long been used for the therapeutic management of human ailments. In this study, we sought to characterize the cytotoxic properties of newly derived purified fractions from previously described camel urine extract (PMF) on various cancer cell lines. Methodology: Two new size dissimilar fractions of PMF (large and small) were obtained by fractionalizing PMF using 3kD and 50kD membrane filters. A SRB cytotoxicity assay of the PMF fractions was performed on cancer cell lines (A549, HCT116, HepG2, MCF-7, U251 and Hela) as well as normal cell lines (human fibroblast cell line and Vero). Results: This study showed that the newly derived and more purified fraction of PMF (new PMF) possesses effective and selective anti-cancer properties against several types of cancer cell lines. Conclusion: This study, as well as previous ones, suggests that camel urine extracts (old and new PMF) may provide newer therapeutic alternatives to clinically manage cancer patients. However, further studies are needed to verify these positive preliminary results.


INTRODUCTION
Historically, it is well known that man has used various parts of animals, including animal urine to treat various human ailments.Animal urine is also a source of therapeutic agents used in modern medicine.As an example, urine derived from pregnant mare's urine is the source of estrogens in a prescription drug used to manage ailments in pre-menopausal and menopausal women [1][2][3][4][5][6].
The successful use of Cisplatin, a platinum complex drug, as a potent anti-tumor agent has triggered interest in other metal-containing compounds as potential anticancer drugs [7].In previous studies, we fractionated camel urine and showed that one fraction (PMF) induced apoptosis and inhibited cell proliferation in various cancer cell lines [8][9][10][11].A follow up study identified the presence of nanoparticles in the PMF fraction; these nanoparticles are composed of cesium and rubidium [12].
Further investigations of PMF compounds is essential to gain a better understanding of their bioactivity and to identify potential anti-cancer compounds that may be used in cancer therapy.The sub-fractionation of PMF according to molecular size using a filtering membrane is one approach for identifying the active components and establishing their efficiency as candidates as cancer drug.In this study, we evaluated the cytotoxic properties of different PMF extracts in the presence or absence of protein macromolecules.To remove any existing macromolecules, the PMF extract was filtered using a 3KD filter tube.For purposes of comparison, the bio-activity tests of the filtrate were carried out under the same experimental conditions as used originally on PMF.The results obtained show that the new PMF extract has effective and selective anti-cancer activity in a range of different cancer cells.

Camel Urine
Camel (Camelus dromedarius) urine was retrieved from female camels kept under hygienic modern husbandry conditions and at the Camel and Range Research Center in Al Jawf, Saudi Arabia.Urine samples were received either fresh or frozen.Samples received were examined microscopically; they were also cultured in MacConkey and blood agars to rule out bacterial infection.

PMF Extraction of Camel Urine and Preparation of its Sub-fractions
The PMF fraction was prepared as previously described [9]; it was then filtered with ethanol using 0.1 or 0.2 µm nylon filters in order to separate the larger from the smaller molecules as sub-fractions of PMF.Two concentrations (100 and 10 µg/ml media) of the stock solution of the filtered PMF were used.Finally, the PMF was sub-fractionated into large and small fractions using a 3 kD centrifuge and 50 kD tubes (Millipore, USA).

Sample Ultra-filtration
To remove glycerol from the filter membrane, the 3 KD centrifugal tube filters were washed several times with 0.5 ml water then centrifuged at 3500 g and 37ºC until no nuclear magnetic resonance (NMR) signal was obtained from the filtrate.The filters were kept moist at 4ºC prior to use, then 0.5 ml samples of urine extract (PMF) were filtered by centrifugation at 10,000 g with the temperature maintained at 4ºC throughout.A volume of 450 µl of the filtrate was transferred to a 5 mm NMR tube followed by the addition of 50 µl of D 2 O.

NMR Spectrometry
The study used NMR spectrometry to identify the small and large molecules in the PMF fraction.

SRB Cytotoxicity Assay
Each of the 8 cell lines were plated with densities of 1x10 4 cells per well, in separate 96-well plates, and incubated at 37°C in a humidified incubator with 5% CO 2 for 24 hr.The180 µl medium was replaced with different concentrations of fractions (10, 7.5, 5, 2.5 and 1 µg/ ml) without disturbing the cells.For each fraction, each concentration was repeated 6 times.Plates were then incubated for 48 or 72 hr at 37ºC in a humidified incubator with 5% CO 2 .In the next step, 50 µl of cold 50% (w/v) TCA was added to each well without removing the medium followed by incubation at 4ºC for 1 hr to fix the cells, rewashing with water 4 times and drying at room temperature.Then, 100 µl of 0.57% (w/v) SRB solution was added to each well and the plates were incubated at room temperature for 30 min.
Using acetic acid (1%;v/v), the washing step was repeated 4 times to remove the unbound SRB dye; the plates were dried at room temperature.Finally, 200 µl of 10mM (w/v) Tris base solution (pH 10.5) was added to each well and the plates which were then incubated for 30 min.Optical density (OD) was measured at 510 nm in a microplate reader and expressed as a mean of the optical density obtained from three independents experiments; each experiment was performed in three replicate wells.

Statistical Analysis
The data obtained were plotted with the drug concentration.Statistical analysis was performed with SPSS and graph pad statistical programs.

Ethical Approval
The study was approved by the Research Ethics committee of the King Abdul Aziz University.

RESULTS
In this study we used proton NMR spectroscopy to evaluate the removal of protein and other macromolecules from fraction of PMF because proton NMR signals of macromolecules are broad compared to the corresponding signals of small molecules.Fig. 1 shows the 600 MHz proton NMR spectrum of camel urine extract and only a few molecules can be identified.Fig. 2 demonstrates the difference between the proton NMR spectra of the extract samples with and without proteins post separation using a 3KD filter tube.As shown in Fig. 3, all the aromatic region signals were identical in both spectra indicating that these signals came from small molecules.Additionally, it confirms that the filtration process was effective in separating macromolecules from small molecules.
Using a number of different cancer cell lines the cytotoxic effects of original PMF (old), the new PMF filtrate (mixture), and its separated parts comprising small molecules (MW less than 3 KD) and the high molecular weight fraction (MW greater than 50 kDa) were tested.The results show that the small molecules fraction of PMF had cytotoxic effects on various cancer cell lines.However, the new PMF mixture was found to have greater cytotoxic effects on various cancer cell lines than did its fractions, small or large molecules.These results suggest that the smaller molecules are the ones with the anticancer properties but that these molecules may depend on macromolecules to increase and facilitate these anti-cancer activities, or possibly to interact with cancer cell receptors.

The effect of PMF fraction on normal skin cells (HFS)
Investigation of the effects of PMF on normal skin cells (HFS) revealed that, even at very high concentrations, PMF (or its fractions) had no significant cytotoxic effects on HFS compared with cancer cells (MCF7, A549, HCT116, U251; HepG2).The results showed slight decrease in the number of cells at the beginning of experiments (<0.025) due to the high concentration of drugs (Fig. 4).Moreover, the results showed nourishing effect of the PMF fraction on normal skin cell HFS with the IC 50 concentration of cancer cells as illustrated in Fig. 5.

The effect of PMF fraction on Vero cell lines
As with normal skin cells (HFS), the results showed that, even at very high concentrations, neither PMF nor its fractions had any significant cytotoxic effect on normal Vero cells as compared with cancer cells (MCF7; A549, HCT116, U251; HepG2).There was a slight decrease (<0.25) in the number of the cells at the beginning of the experiments, this being due to the high concentration of drugs present at (Fig. 6).

The effect of different PMF fractions on cancer cells
Several experiments were conducted to compare the cytotoxic effects of the purified PMF extracts on cancer cell lines with those of the parent extracts.The results obtained indicated that the effect of the new fraction with extra purification (new PMF, which consists of both large and small molecules) is greater than that of the parent extracts, as shown in the next experiments.

The effect on lung cancer cells (A549)
These parts of the experiments were preformed to compare the effects of the old PMF fraction (G), the new PMF fraction (mix), and the parent extracts of PMF, comprising only small (S) or large molecules (L) on lung cancer cells (A549).The results showed that the new PMF fraction (mix) had a stronger cytotoxic effect on A549 cells than did the old PMF fraction or its component parts (small or large molecules) (Fig. 7).

The effect on breast cancer cells (MCF7)
These experiments were designed to compare the effects of the old PMF fraction (G), new PMF (mix), and the separated large (L) and small molecules (S) of PMF on breast cancer cells (MCF-7).The obtained results showed that the new PMF fraction (mix) had a more cytotoxic effect on the MCF-7 cells than the old fraction (G), or its component parts, the small (S) and large (L) molecule fractions (Fig. 8).

The effect on cervical cancer (Hela) cells
The results from this experiment indicated that the new PMF fraction had a greater cytotoxic effect on Hela cells than its component small (S) and large (L) molecule sub-fractions (Fig. 9).

The effect on human glioma cell lines U251 (glial)
The results showed that the new PMF fraction (mix) had a greater cytotoxic effect on glial cancer cells than did the separated parts of it, the small (S) and large molecule (L) sub-fractions (Fig. 10).

The effect on colon cancer cell line (HCT116)
The results indicated that the new PMF fraction (mix) has greater cytotoxic effects on colon cancer cells than the separated fractions of it, the small (S) and large (L) molecules (Fig. 11).

The effect on Human hepatocellular liver carcinoma cell line (HepG2)
The results showed similarly that the cytotoxic effect of the new PMF fraction (mix) on liver cancer cells was greater than the effects of its separated component sub-fractions, the small (S) and large (L) molecules (Fig. 12).

DISCUSSION
Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool allowing investigation of the chemical composition of a given sample [14-16].Furthermore, NMR can be used for structural elucidation and to study molecular dynamics [17][18][19][20][21][22].Consequently, NMR is generally used as a complementary technique to mass spectrometry (MS) for the identification and quantitative analysis of the chemical composition of natural products and biological samples, such as urine [12,[23][24][25][26].In fact, both NMR and MS have been used intensively for drug discovery from natural products [27,28], for drug assessment [29], for characterizing newly designed drugs [30,31], and for evaluating drug toxicity [32].Wishart et al. for example, employed NMR spectroscopy in conjunction with different MS techniques to establish that 209 different metabolites can be identified in human urine using NMR [33].However, the inherent limitation of NMR spectroscopy is a low sensitivity meaning that compounds present at a concentration lower than micromolar cannot be detected.However, developments in NMR machinery, such as the use of cryogenic probes [34], micro-probes [35] and increased magnetic field strength [36], as well as the invention of dynamic nuclear polarization (DNP) [37][38][39][40], continue to enhance the applicability of NMR approaches.
The cytotoxic properties of the bioactive fraction (G) of camel urine (PMF) against different types of cancer cells have been studied previously [41,42]: these studies showed that, although PMF was highly cytotoxic against A549 human lung cancer cells and leukemic L1210 cells, it did not harm normal human cells; this indicates that PMF can be used to selectively target cancer cells.The results of the current study of the cytotoxic effects of purified fractions of PMF on different types of cancer cell lines are in accord with those of previous studies [43][44][45][46][47] reporting the cytotoxic effect of PM701 (lyophilized camel urine) on cancer cell lines such as A549, L1210, HCT116, HepG2, MCF-7 and U251.Moreover, other fractions of camel urine such as PM701 and its bioactive fraction PMF and sub-fraction PMFK were shown to have cytotoxic properties against different cancer cell lines.In another study, the addition of PMF to A549 cells for different periods of treatment caused a series of changes in the A549 cells; live images of these cells showed that the severe lethal effects of PM701 on them began immediately after addition of the substrate [45].It was established that the nuclei of cancer cells incubated in PM701 is attacked by the substrate, this leading to an irreversible degradation of the cancer cells.
As with the current study, a previous study [41][42][43][44][45][46] demonstrated that PM701, its bioactive fraction (PMF), and its sub-fraction (PMFK) all exhibit anti-cancer activity.Another study examined the effect of lyophilized PM701 and its bioactive fractions on the growth of breast cancer cells (MCF-7).PM701, PMF and PMFK were shown to significantly inhibit the proliferation of MCF-7 cells, demonstrating the apoptotic effect of PM701 and its fractions on breast cancer cells, through its direct action on the cell nuclei [11].In more deep studies, we have shown that PM701 is natural, easily available, cheap, sterile, nontoxic, and causes selective cell death of cancer cells.
Unlike several anti-cancer chemotherapeutic agents, PM701 did not require high concentrations to be effective.In fact, PM701 has been found to have nourishing effects on normal skin fibroblasts in a tissue culture medium.The effect of PM701 in A549, L1210 and on other cancer cell lines is well documented [41,42,46,47].
The results obtained here are consistent with preclinical studies [44] that demonstrated that PM701 has no hepatotoxic, nephrotoxic effects, nor any hematological toxicity.Histopathological preclinical studies showed that PM701 has no effects on the gastric mucosa and that it does not alter the parenchyamatous architecture of the liver and kidneys, which display a preserved hepatocellular outline and no signs of necrosis.Moreover, there was found to be an enlargement of the germinal centers of the white pulp lymph nodules in the spleen, indicating the activation of the immune system without any concomitant effect on the vital body organs.
Finally, under all experimental conditions used, camel urine extract (PMF) has been established to be non-toxic and no significant adverse effects have been observed.This natural product, which has been used for many years for the treatment of a range of human diseases and without any reported harmful effects, is of considerable interest on the basis of its anticancer activity.In fact, early Phase I clinical trials using capsules and syrup containing PMF have been carried out on healthy volunteers to confirm that no harmful side effects were caused [43].Further work on PMF is required and this agent remains under intensive investigation.

CONCLUSION
In this study, the anti-cancer properties of two fractions of PMF (formed by separation of the small molecules from the macromolecules using 3kD and 50kD membrane filters) were evaluated.
It was established that the optimum anti-cancer effects of PMF are obtained using the mixture of both small and large molecules.These results suggest that the bioactive molecules are the smaller ones, while the large molecules may work as receptors and transport the bioactive molecules into the cancer cells.These results combined with previous reports indicate that Camel urine and its bioactive fraction PMF are potential anti-cancer drugs with negligible effects on cells derived from vital organs such as the liver and kidney.It is suggested that PMF be subjected to further studies including clinical trials.

Fig. 1 .Fig. 2 .Fig. 3 .
Fig. 1.The 600MH Z 1 HNMR spectrum of original camel urine extract in D 2 O at 298K shows that the PMF extract contains both small molecules and macromolecules (mainly proteins), where the broadened peaks (black arrows) indicate typical protein signals