|Year : 2018 | Volume
| Issue : 1 | Page : 53-58
Studies on 3H-levamisole binding to murine splenic lymphocytes, normal, malignant human lymphocytes and fate of levamisole in cell culture
B Nageshwari1, Ramchander Merugu2
1 Department of Biotechnology, Government College Autonomous, Rajamahendravaram, Andhra Pradesh, India
2 Department of Biochemistry, Mahatma Gandhi University, Nalgonda, Telangana, India
|Date of Web Publication||5-Mar-2018|
Dr. B Nageshwari
Department of Biotechnology, Government College Autonomous, Rajamahendravaram, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: Levamisole at high concentrations has been shown to have anticancer and immunosuppressive actions. Methods: In the present study, fate of levamisole in cell culture and 3 H-levamisole binding to murine splenic lymphocytes, normal and malignant human lymphocytes has been investigated. Results: High-performance liquid chromatography analysis of cell culture supernatants of myeloma cells treated with levamisole has shown that products of levamisole appeared with progressive culture period indicating a metabolic transformation. 3 H-levamisole binding assays indicate that the binding was signifi cantly higher in lysates of lipopolysaccharide-stimulated murine splenic lymphocytes as compared to whole cells. Conclusion: The degradation of levamisole could be one more possible reason for the high concentration of levamisole required to get the desirable cytotoxic effect on myeloma cells.
Keywords: High-performance liquid chromatography analysis, levamisole, lymphocytes, myeloma cells
|How to cite this article:|
Nageshwari B, Merugu R. Studies on 3H-levamisole binding to murine splenic lymphocytes, normal, malignant human lymphocytes and fate of levamisole in cell culture. Biomed Biotechnol Res J 2018;2:53-8
|How to cite this URL:|
Nageshwari B, Merugu R. Studies on 3H-levamisole binding to murine splenic lymphocytes, normal, malignant human lymphocytes and fate of levamisole in cell culture. Biomed Biotechnol Res J [serial online] 2018 [cited 2020 Apr 3];2:53-8. Available from: http://www.bmbtrj.org/text.asp?2018/2/1/53/226573
| Introduction|| |
Levamisole is an antihelminthic drug and has also been used as an immunomodulator in conjunction with 5-Fluorouracil as an adjuvant in the treatment of colon cancer relapse., At high concentrations, levamisole has been shown to have anticancer and immunosuppressive actions. Studies from our laboratory have shown that lipopolysaccharide (LPS) stimulated murine B-lymphocytes secrete high levels of Alkaline phosphatase (APase) and that levamisole inhibits the proliferative response and APase activity in a dose-dependent manner. High levels of APase activity has been observed in different types of malignant cells and serum samples of cancer patients., In earlier studies, the effect of levamisole on myeloma cells has been observed to be dose-dependent, but the high concentration of levamisole needed to bring about the observed effect is not known.
Levamisole, being a freely water-soluble compound, it is expected that its entry into the cells through the lipid bilayer will be meager. Levamisole has been shown to be decomposed enzymatically/non-enzymatically into three degradation products (Kimberly et al. 1991). The structures of the purified products are (a) 3-(2-mercaptoethyl)-5-phenyl-imidazolidine-2-one (b) 6-phenyl-2,3-dihydroimidazo (2,1-b) thiazole and (c) bis (3-(2-oxo-5 phenyl imidazolidine-1-yl) ethyl) disulfide. The decomposition of levamisole has been shown to be temperature and pH dependent. Hence, the free and unaltered levamisole concentration available for interaction with cells could be very low as compared to the final concentration in cell culture as a result of protein binding and degradation. Apart from decomposition, other reason for the high concentration of levamisole needed in cell culture to get the effects could be due to low binding of levamisole to the cells. Hence, in this paper, it is attempted to determine the fate of levamisole in cell culture by estimating the concentration using high-performance liquid chromatography (HPLC) at different culture time periods. Further mitogen-stimulated murine splenic lymphocytes, myeloma cells and normal human peripheral blood cells were assayed for APase activity, and the interaction of levamisole with cells was studied by estimating the binding of 3H-levamisole to whole cells and broken cell preparations.
| Methods|| |
Alkaline phosphatase assay
APase activity was determined by p-nitrophenol (p-NPP) hydrolysis. The absorbance of p-NP produced was measured at 405 nm. Cells were dispensed into wells of microliter plate and centrifuged at 450 g for 10 min at 4°C. The cells were suspended in 0.9% saline, and the centrifugation step was repeated. To the pellet 180 μl of 1 mg/ml p-NPP in 0.1M bicarbonate buffer with 2 mM MgCl2 was added. Incubation was carried out at 37°C in a humidified incubator. After 30 min, the reaction was stopped by the addition of 20 μl of 1N NaOH and the absorbance was measured at 405 nm using ELISA plate reader. The amount of p-NP released was calculated from a standard graph. The results were expressed as nmoles of p-NP released/0.2 × 106 cells.
Analysis of levamisole by high-performance liquid chromatography
Cell culture and extraction of levamisole
Myeloma cells, 0.25 × 106/ml were cultured with and without FCS and levamisole was added at a concentration of 2.5 mM. At 0, 24, 48, 72 h of culture period, 1 ml cell suspension was taken and centrifuged at 500 g, and the supernatant was collected. Levamisole was extracted according to the protocol described earlier., Briefly, one ml culture supernatant was taken in a 15 ml polypropylene centrifuge tube, and 0.8 ml water was added and vortex mixed. Then, 0.5 ml of 10N sodium hydroxide was added and vortex mixed. To this, 5 ml of ethyl ether: N-hexane, 80:20 (v/v) was added and vigorously shaken. The mixture was centrifuged for 5 min at 850 g and the organic layer was separated and dried at room temperature under a stream of nitrogen. The residue was redissolved in 1 ml of mobile phase and filtered through a 0.22 μm pore-sized filter and 20 μl was used for HPLC analysis.
High-performance liquid chromatography analysis
A stock solution of 1 mg/ml levamisole was prepared in methanol. It was stored at − 20°C and used. An HPLC system (Waters, USA) fitted with a 5 μm C18(Octadecylsilane), 150 mm × 4.6 mm analytical column (Phenomenex, USA), guard column packed with Perisorb RP-18 (Upchurch Scientific, USA) and with an ultraviolet (UV)-detector was used. The chromatography was carried out using the following conditions:
- Elution: Isocratic, sample volume: 20ul, Flow rate: 1 ml/min
- Mobile phase: 2% Acetic acid in water, methanol (50:50(v/v)) pH adjusted to 7.30 with 10N NaOH solution
- Detection: UV detection at a wavelength of 225 nm.
Levamisole, 5–20 μg was analyzed as standard along with the extracted samples. The concentration in the samples was determined using a graph obtained with values of standard levamisole.
Resting and mitogen-stimulated murine and human peripheral blood lymphocytes and myeloma cells were used for binding studies.
3H-levamisole binding to intact cells
1 × 106 cells were taken and washed twice with RPMI-1640 and then resuspended in 100 μl of RPMI-1640, H-levamisole (50 nmoles, 135,000 cpm) was added and incubated at 37°C for 1 h. At the end of incubation, the cells were washed twice with RPMI-1640, and finally, the cell pellet was lysed using 100 μl of 0.1% TX-100 and counted in Bray's mixture.
3H-levamisole binding to lysate
1 × 106 cells were taken and washed twice with RPMI-1640 the cell pellet was then lysed with 0.1% Triton X-100. To 0.10 ml of the cell lysate, H-Levamisole (50 nmoles, 135,000 cpm) was added and incubated for 1 h at 37°C. Then, 10% trichloroacetic acid (TCA) was added to a final concentration of 10% and incubated for 1 h at 4°C. The precipitate was pelleted by centrifugation at 5000 g for 10 min, and the pellet was washed twice with 5% TCA and finally with ether. The pellet was air-dried and dissolved 100 μl of 0.1% TX-100 and counted in Bray's mixture. Nonspecific binding was determined in the presence of a 1000-fold molar excess of unlabeled compound. Nonspecific binding was subtracted from the total to obtain the specific binding.
| Results|| |
Analysis of levamisole by high-performance liquid chromatography
Levamisole eluted with a retention time of about 6.0 min on reverse phase column under the conditions employed. Levamisole was extracted from myeloma cell cultures at various time points 0, 24, 48, 72 h and analyzed by HPLC. The elution profiles of levamisole at various time periods of culture are presented in the [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d and [Table 1]. The elution profiles indicated that compounds with retention times different from that of levamisole were obtained with progressive culture time period. The amount of levamisole recovered from the culture supernatants also decreased with time.
|Figure 1: (a) high-performance liquid chromatography elution profile of levamisole extracted from culture – “0” time of culture period. (b) High-performance liquid chromatography elution profile of levamisole extracted from culture – 24 h of culture period. (c) High-performance liquid chromatography elution profile of levamisole extracted from culture – 48 h of culture period. (d) High-performance liquid chromatography elution profile of levamisole extracted from culture – 72 h of culture period|
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|Table 1: Levamisole extracted at various time points and analyzed/quantified by high-pressure liquid chromatographic|
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Tritium-labeled levamisole binding assays were performed using unstimulated and mitogen-stimulated murine splenic lymphocytes and human peripheral blood lymphocytes. Earlier studies have shown that mitogen-stimulated murine splenic lymphocytes show enhanced APase activity. This experimental system was used for comparative purposes to assess the binding of 3H-levamisole to myeloma cell lines.
LPS was used for mitogenic stimulation in case of murine splenic lymphocytes, and PWM was used for human peripheral blood lymphocytes. LPS stimulated murine splenic lymphocytes showed enhanced APase activity [Figure 2]a. No enhancement of APase activity was observed in human peripheral blood lymphocytes upon mitogenic stimulation [Figure 3]a. However, myeloma cell lines displayed significant APase activity [Figure 4]a.
|Figure 2: Alkaline phosphatase activity and 3H-Levamisole binding of mitogen stimulated murine splenic lymphocytes. (a) *P < 0.05 control versus mitogen. (b) 1-3: Whole cells, 4-6: Cell lysate. *P < 0.05 lysate versus whole cells|
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|Figure 3: (a) Alkaline phosphatase activity and 3 H--Levamisole binding of poke weed mitogen stimulated normal human peripheral blood cells. (b) 1, 2: Whole cells, 3, 4: Cell lysate. *P < 0.05 lysate versus whole cells|
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|Figure 4: Alkaline phosphatase activity and 3 H--Levamisole binding of myeloma cell lines. (b) 1, 2: Whole cells, 3, 4: Cell lysate. *P < 0.05 lysate versus whole cells|
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The binding of 3H-levamisole was significantly higher in lysates of LPS stimulated murine splenic lymphocytes as compared to whole cells [Figure 2]b. The binding of 3H-levamisole to human PBL was minimal in whole cells as well as in lysates, and there was no difference between unstimulated, and PWM stimulated cells [Figure 3]b. The myeloma cells which express APase activity had a significant 3H-levamisole binding [Figure 4]b. In all the cases, H-levamisole binding correlated well with the expression of APase activity.
| Discussion|| |
Previous studies have revealed that levamisole inhibits proliferative response of multiple myeloma cells probably through inhibition of APase activity. The concentration of the drug required to get half-maximal inhibition in myeloma cells was shown to be around 1 mM. This corresponds to a value of 240 μg/ml. Drugs are transported mostly as complexes with serum albumin. The binding of levamisole to total plasma proteins of 6 animal species was determinedin vitro by equilibrium dialysis, and it has been shown to bind avidly to plasma proteins. Levamisole being a basic organic drug has a weak lipophilic tendency in an alkaline medium. It was reported earlier that levamisole when administered to patients with colorectal carcinoma at a concentration of 100 mg/m 2 three times a day, resulted in 1 μg/ml of peak plasma concentration. The high concentration required in cell culture could be first due to low lipophilic nature of levamisole thereby resulting in low binding to membrane proteins and second due to binding to serum proteins resulting in lower concentration of free drug available to the cells. It is also possible that the drug undergoes transformation under the culture conditions employed.
To address this issue, H-levamisole was used to estimate its binding to cells. Earlier, H-labeled levamisole specific binding assays revealed a specific binding carrier for levamisole in human peripheral lymphocytes and granulocytes. The results from the present study of 3H-levamisole binding assays indicate that the binding was significantly higher in lysates of LPS stimulated murine splenic lymphocytes as compared to whole cells. The binding of 3H-levamisole to human PBL was minimal in whole cells as well as in lysates, and there was no difference between unstimulated, and PWM stimulated cells. The myeloma cells which express APase activity had significant 3H-levamisole binding.
| Conclusion|| |
In all the cases, H-levamisole binding correlated well with the cells expressing APase activity. Enhanced binding in the lysates compared to whole cells could be due to the exposure of putative levamisole binding site (domain) of APase present on cell membrane facing the cytosol.
Earlier studies have reported the effect of temperature and pH on the chemical stability of levamisole where levamisole was shown to degrade rapidly between pH 5–8., Levamisole solution stored at 4°C was shown to be stable. Levamisole stability was assessed when stored at 4 or 37°C and pH 6, 7, 7.5, and 8. Analysis of the various solutions by high-pressure liquid chromatography demonstrated that levamisole degradation occurs during storage in neutral and alkaline conditions to form three products. The formation of the products was accelerated by increasing the temp from 4 to 37°C. The degradation products were purified by preparative high-pressure liquid chromatography and their structures determined by spectrometry, IR spectrometry, and homo- and hetero-nuclear two-dimensional NMR spectroscopy.
Levamisole, used presently in the culture along with cells, medium (RPMI 1640) and fetal calf serum at 37°C up to a period of 72 h might have affected the stability of levamisole, to assess the same HPLC was done. A HPLC-UV detection method was used for quantification of levamisole. Calibration curves for levamisole were linear over the range 4–20 ug. HPLC analysis of cell culture supernatants of myeloma cells has shown that products of levamisole appeared with progressive culture period indicating a metabolic transformation. Furthermore, the amount of levamisole recovered from the culture supernatants with FCS was lower (less stable) than levamisole extracted from culture samples without FCS. This could be due to additional enzymatic degradation of levamisole in the presence of serum apart from degradation due to temperature. The degradation of levamisole could be one more possible reason for the high concentration of levamisole required to get the desirable cytotoxic effect on myeloma cells.
We would like to thank Janssen Research Foundation-Belgium for providing us with 3H-levamisole (gratis) to carry out a part of the work embodied in this paper.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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