Accepted Manuscript

Differential Effects of Anti-cancer and Anti-hepatitis drugs on liver cystatin

Aaliya Shah, Medha Priyadarshini, Mohd Shahnawaz Khan, Mohd Aatif,Fakhra Amin, Shams Tabrez, Galila F Zaher, Bilqees Bano

PII: S1319-562X(14)00073-4DOI: http://dx.doi.org/10.1016/j.sjbs.2014.06.006Reference: SJBS 317

To appear in: Saudi Journal of Biological Sciences

Received Date: 27 April 2014Revised Date: 27 June 2014Accepted Date: 28 June 2014

Please cite this article as: A. Shah, M. Priyadarshini, M.S. Khan, M. Aatif, F. Amin, S. Tabrez, G.F. Zaher, B. Bano,Differential Effects of Anti-cancer and Anti-hepatitis drugs on liver cystatin, Saudi Journal of BiologicalSciences (2014), doi: http://dx.doi.org/10.1016/j.sjbs.2014.06.006

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Differential Effects of Anti-cancer and Anti-hepatitis drugs on liver

cystatin

Aaliya Shah1, Medha Priyadarshini2, Mohd Shahnawaz Khan3, Mohd Aatif1, Fakhra

Amin1, Shams Tabrez4, Galila F Zaher5, and Bilqees Bano*1

1Department of Biochemistry, F/O Life Sciences, AMU, Aligarh, INDIA.

2Department of Medicine, Division of Endocrinology, Metabolism and Molecular

Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611,

USA

3Department of Biochemistry, College of Science, King Saud University, KSA

4King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi

Arabia

5Department of Haematology, College of Medicine, King Abdulaziz University, Jeddah

21589, Saudi Arabia

Corresponding Author:

Professor Bilqees Bano

Department of Biochemistry, F/O Life Sciences,

Aligarh Muslim University, INDIA

Email: bbano08@rediffmail.com

ABSTRACT

The drug-protein interaction has been the subject of increasing interest over the decades.

In the present communication, the interaction of liver cystatin with anti-cancer

(adriamycin) and anti-hepatitis (adevofir dipivoxal) drug was studied by thiol-protease

inhibitory assay, UV absorption, fluorescence spectroscopy and Circular dichroism (CD).

A static type of quenching was observed between the protein and the drug molecules.

Binding constant (Ka) of adriamycin to liver cystatin (LC) was found to be 1.08x106M-1.

Moreover, binding sites number was found to be 2. Importantly, cystatin loses its activity

in the presence of adriamycin. However, intrinsic fluorescence studies in the presence of

adevofir dipivoxal showed enhancement in the fluorescence intensity suggesting that

binding of adevofir to LC caused unfolding of the protein. The unfolding of the test

protein was also accompanied by significant loss of inhibitory activity. CD spectroscopy

result showed, both adriamycin and adevofir dipivoxal caused perturbation in the

secondary structure of liver cystatin. The possible implications of these results will help

in combating drug induced off target effects.

Keywords: Liver cystatin, Adriamycin, Adevofir dipivoxial, Fluorescence, UV-

spectroscopy

1. INTRODUCTION

Drug-protein associations are vital, since most of the administered drugs are reversibly

bound to proteins. The bound drugs are transported mainly as a complex with these

proteins. The binding factors are useful in studying the pharmacological response and

drugs dosage design (Borga and Borga, 1997). The present report summarizes the

interaction of goat liver cystatin (thiol-proteinase inhibitor) with an anti-sarcoma drug,

Adriamycin and an anti-hepatitis drug, Adevofir dipivoxil. Adriamycin (doxorubicin

hydrochloride) is an excellent anti-tumor antibiotic and is very effective against a large

number of human malignancies. The anti-cancer activity of adriamycin is associated with

the formation of intercalative complexes with DNA (Bryn and Dolch, 1978).

Adevofir dipivoxil is a diester prodrug of adevofir. It is an acyclic nucleotide analogue

having activity against human hepatitis B virus (HBV). Moreover, it inhibits HBV-DNA

polymerase (reverse transcriptase) action via natural substrate deoxyadenosine

triphosphate binding and DNA chain termination. The chemical structure of Adriamycin

(Doxorubicin Hydrochloride) and Adevofir dipivoxil is shown in figure 1 and 2

respectively.

Cystatins are the family of proteins that regulate and inhibit the detrimental effect

associated with cysteine proteinases (Ekiel et al., 1997). Cystatins could protect the cells

from unnecessary proteolysis which might leads to several pathological conditions (Shah

and Bano, 2009).

The goat liver cystatin used in the present study was purified in our laboratory (Shah and

Bano, 2011). Further, conformational changes in the purified thiol protease inhibitor

after association with anti-cancer and anti hepatitis drug were monitored by UV-visible,

fluorescence and circular dichroism spectroscopic techniques. Moreover, the current

paper also addresses the kind of interaction involved in the binding of these drugs with

thiol protease inhibitor.

2. MATERIALS AND METHODS

MATERIALS:

Casein, papain, EDTA, acetone, sephacryl-S100HR, CBB R-250 and cysteine were

procured from Sigma Aldrich. Adriamycin (Doxorubucin hydrochloride) was purchased

from VHB Life Sciences Limited India. Adevofir dipivoxil was purchased from Sun

Pharmaceutical Industries, India. All other chemicals used were of analytical grade.

METHODS:

2.1.Protein Estimation

The concentration of purified protein was quantitated by Lowry et al. (1951) method.

2.2.Preparation of Drug Solutions

As adriamycin (ADR) is sensitive to light and oxygen, a stock solution of ADR within

the theraupetic range in normal saline was prepared just before use. 2µM of goat LC was

incubated with varying concentrations of ADR in the range of 0.5-3µM for 30 minutes.

Moreover, a stock solution of adevofir dipivoxil in 0.05M sodium phosphate buffer (pH

7.2) was prepared fresh just before use. Goat liver cystatin at a concentration of 2 µM

was incubated with varying concentration of adevofir dipivoxil (0.1 µM-1 µM) for 30

minutes.

2.3. Thiol proteinase inhibitory activity assay

Aliquots from the incubated samples were tested for their thiol protease inhibition

potential by the method of Kunitz (1947).

2.4.UV-Visible spectroscopy

Absorption spectra of cystatin and cystatins incubated with ADR and adevofir dipivoxil

were measured on UV-visible spectrophotometer at 220-400 nm wavelength range by the

use of 1cm path length cell holder.

2.5.Fluorescence spectroscopy

The measurements of fluorescence were performed on a spectrofluorometre (Shimadzu)

at 25˚C by the use of a quartz cell of 1cm path length. The fluorescence of cystatin bound

drugs were recorded at the wavelength range of 250-400 nm after exciting the complex at

280 nm.

2.6.Circular dichroism measurement

Far-UV CD measurements were performed by use of a circular dichroismchiroptical

spectrometer (Applied Photophysics, Chira-scan-Plus, UK). Samples were maintained at

250C with the help of circulating water bath in a 1 mm quartz cuvette. Spectra of LC in

the absence and presence of various concentrations of adriamycin and adevofir dipioxal

were measured in the range 190-250 nm with a step size of 1.0 nm.

RESULTS AND DISCUSSION

2.7.Interaction of liver cystatin with adriamycin

Fluorescence measurements reveal information about the binding of small molecules with

proteins, such as binding constant, binding sites and binding mechanism. Binding of

ADR with goat liver cystatin caused quenching in the fluorescence intensity. The

concentration increase of ADR resulted in the rise in quenching of the cystatin-ADR

complex. The fluorescence emission spectra of the said complex in the presence of

increasing concentration of ADR have been illustrated in Figure (3). The maximum

quenching was observed at 3µM adriamycin concentration. To determine the mechanism

of binding between ADR and goat liver cystatin the fluorescence intensity data were

analyzed by Stern-Volmer equation (Shang et al., 2006).

Literature analysis illustrates two type of quenching namely static and dynamic. Static

quenching involves the formation of a stable complex between the fluor and quencher.

On the other hand, in dynamic quenching the ligandhits with excited fluor, leads to loss

of some energy.

The plot of F₀ /F vs [Q] exhibited a good linear relationship indicating, the interaction

was purely static in nature (Figure 4).

The binding constant and the number of binding sites can be determined by the equation

given by Gao et al. (2004).

Log [(F₀-F)/F] =Log K n Log [Q].

Where K and n are the binding constant and binding site numbers. Binding constant was

found to be 1.08×106M-1 and the binding sites number was found to be 2 as shown in

figure 5.

2.8.UV- visible spectra of adriamycin cystatin complex

Absorption spectral measurements on liver cystatin in the presence of drugs provided

information related to their interaction. Difference spectra of drug protein complex were

measured against protein alone (Figure 6). For the difference spectra obtained at 0.1µM

ADR, positive peaks at 260 nm might have the contribution of phenylalanine. The

negative peak at 210 nm observed for liver cystatin-ADR (LC-ADR) complexes at 1, 2

and 3µM ADR concentrations respectively may have contributions from histidine

residues (Donovan, 1969). The intense negative peak at 260 nm for LC-ADR complexes

is indicative of involvement of phenylalanine and tyrosine in complexation process. The

broad shoulders at 290 nm are also due to tryptophan and may contain contribution from

phenyalanine (Gao et al., 2004).

2.9. Inhibitory activity of adriamycin cystatin complex by papain

Changes in the inhibitory activity of LC after incubating for 30 minutes with increasing

concentration of LC are shown in the Table 1. The results show that liver cystatin lost

complete inhibitory activity at 3µM concentration of adriamycin. This suggests that

increasing concentration of adriamycin resulted in the functional inactivation of cystatin.

2.10. Fluorescence spectra of adevofir dipivoxal cystatin complex

Binding of adevofir dipivoxal with goat liver cystatin led to an increase in the

fluorescence intensity of the goat liver cystatin indicating that the binding caused

unfolding of the protein as shown in figure 7. Maximum unfolding was observed at 1µM

concentration of the drug. Increase in fluorescence intensity was also accompanied by a

red shift of 5nm which indicates perturbation in the environment of aromatic residues and

unfolding of goat liver cystatin in the presence of adefovir dipivoxal.

2.11. UV-visible absorption spectra of adevofir dipivoxal cystatin complex

UV-visible absorption difference spectra were computed at varying drug concentrations

from 0.1µM to 1µM. However, profound changes were noted only for those obtained at

0.1µM, 0.5 µM and 1µM concentrations of drug. A sharp negative peak noticeable at 210

nm in difference spectra obtained at 0.1µM adevofir dipivoxal, suggest changes around

the histidine residues. A negative peak noticeable at 280 nm suggests changes around

tyrosine residues (Donovan, 1969). Difference spectra of drug protein complex at 1µM

drug concentration showed broad shoulder at 260 nm, indicative of involvement of

phenylalanine (Figure 8).

2.12. Inhibitory activity of goat liver cystatin in the presence adevofir dipivoxal

Changes in the inhibitory activity of goat liver cystatin with increasing concentration of

adevofir dipovixal are shown in Table 2. The results show that goat liver cystatin lost

significant amount of inhibitory activity at 1µM concentration of adevofir dipivoxal. Loss

of inhibitory activity could be attributed to the modulation in the conformation of goat

liver cystatin.

2.13. Drug-Protein interaction analysis: Circular dichroism measurement

Circular Dichroism (CD) is a spectroscopic technique widely used for the evaluation of

the conformation and stability of proteins in several environmental conditions and in the

presence of various ligands. The obtained data showed negative peak around 222 and 208

nm, indicating α-helical characteristic of liver cystatin. While interaction with adriamycin

and adevofir dipivoxal, ellipticity decreases with increasing concentration of drugs

(Figure 9a and 9b). Secondary structural change in the liver cystatin was found to be

more pronounced in the presence of anti-hepatitis drug (Adevofir dipioxal) compared

with anti-cancer drug (Adriamycin).

3. Conclusion

The conformational changes induced in goat liver cystatin upon binding of adriamycin

and adevofir dipivoxal helps in addressing the kind of interactions involved in the

binding. Understanding the molecular basis of these interactions will help in combating

drug induced off target effects which in the present case might be activation or

dsyregulation of cysteine proteases.

Acknowledgment: The authors extend their appreciation to the Deanship of Scientific

Research at KSU for funding this work through research group project number RGP-

VPP-215.

Abbreviations:

LC: Liver cystatin

ADR: Adriamycin

CD: Circular dichroism

Ka: Binding constant

HBV: Human hepatitis B virus

REFERENCES

Borga, O., Borga, B., 1997. Serum protein binding of nonsteroidal antiinflammatory

drugs: a comparative study. J. Pharmacok. Biopharm. 25, 63-67.

Bryn, S.R., Dolch G.D., 1978. Analysis of binding of daunorubicin and doxorubicin to

DNA using computerized curve-fitting procedures. J. Pharm Sci., 67, 688-690.

Donovan, J.W., 1969. Changes in ultraviolet absorption produced by alteration of protein

conformation. J. Biol Chem. 244, 1961-1967.

Ekiel I., Abrahamson M., Fulton D.B., Lindahl, P.1997. NMR structural studies of human

cystatin C dimers and monomers. J Mol Biol.271, 266-271.

Gao, H., Lei, L., Liu, J., Qin, K., Chen, X., Hu, Z. J., 2004. Photochem Photobiol Part

A.167: 213-221.

Kunitz, M., 1947. Crystalline soybean trypsin inhibitor: II. General Properties. J. Physiol.

30, 291-310.

Lowry, H., Rosebrough N.J, Farr A.L., Randall, R., 1951. Protein measurement with the

Folin phenol reagent. J. Biol Chem.193: 265-275.

Shah, A., and Bano, B., 2011. Spectroscopic studies on the interaction of bilirubin with

liver cystatin. European Biophysics Journal. 40, 175-180.

Shah, A., Bano B., 2009. Cystatins in health and diseases. Int. J. Pept. Res.Ther. 15, 43-

48.

Shang, L., Jiang., X.U., Dong, S.J., 2006. In vitro study on the binding of neutral red to

bovine serum albumin by molecular spectroscopy. J Photochem Photobiol A Chem. 184,

93-97.

Thipperudrappa, J., Biradar, D.S., Lagare, M.T., 2006. Fluorescence quenching of BPBD

by aniline in benzene–acetonitrile mixtures.J Photochem Photobiol AChem. 177, 89-93.

Legends to Figures:

1) Chemical structure of Adriamycin (Doxorubicin Hydrochloride)

2) Chemical structure of Adevofir Dipivoxal.

3) Fluorescence emission spectra of adriamycin–cystatin complex in the presence of

differentconcentrations of adriamycin obtained in sodium phosphate buffer, pH,

7.5. Protein concentration was 2µM.Concentration of Adriamycin was (from

bottom to top) 3 µM, 2µM, 1 µM, 0.1µM respectively.

4) Determination of types of quenching by Stern-Volmer constant.

5) Determination of Binding Site by Stern-Volmer.

6) Light absorption spectra of adriamycin–cystatin complex in the presence of

different concentrations of adriamycin obtained in sodium phosphate buffer, pH

7.5. Protein concentration was 1µM.Concentration of 3µM, 2µM, 1 µM, 0.1µM

respectively.

7) Fluorescence emission spectra of adevofir dipivoxal–cystatin complex in the

presence of different concentrations of adevofir dipivoxal obtained in

sodium phosphate buffer, pH7.5. Protein concentration was 1µM.Concentration

of adevofir dipivoxal was (from bottom to top) 0.1µM, 0.5 µM, and 1µM.

8) Light absorption spectra of adevofir dipivoxal-cystatin complex in the presence of

different concentrations of adevofir dipivoxal obtained in sodium phosphate

buffer, pH 7.5. Protein concentration was 1µM. Concentration of adevofir

dipivoxal was (from bottom to top) 0.1µM, 0.5 µM,1µM.

9) Circular dichroism spectra of liver cystatin in the absence and presence of various

concentration of adriamycin (a) and adevofir dipioxal (b). The concentration of

native liver cystatin was (0.2mg/ml).

Tables Legends:

Table 1: Antiproteolytic activity of liver cystatin in the presence of varying concentration

of adriamycin (ADR) after incubation for 30 minutes.

Table 2: Effect of Adefovir dipivoxal on inhibitory activity of liver cystatin after

incubation for 30 minutes

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9a

Figure 9b

TABLE 1:

ANTIPROTEOLYTIC ACTIVITY OF LIVER CYSTATIN IN THE

PRESENCE OF VARYING CONCENTRATION OF ADRIAMYCIN

(ADR) AFTER INCUBATION FOR 30 minutes.

Concentration

of ADR(µM)

% remaining

inhibitory activity

% loss of

inhibitory activity

0 100 0

0.1 80.2±2.5 19

1 74±1.4 26

2 58.2±2.2 41.8

3 ND 100

• The inhibitory activity of LC-I in the presence of ADR was assessed by its ability

to inhibit caseinolytic activity of papain as described by Kunitz.

• ND None detected.

• *The inhibitory activity of the native liver cystatin (LC) was taken as 100.

• Concentration of LC was 1µM

TABLE 2:

EFFECT OF ADEFOVIR DIPIVOXAL ON INHIBITORY ACTIVITY

OF LIVER CYSTATIN (LC) AFTER INCUBATION FOR 30

MINUTES.

Concentration of

Adevofir

Dipivoxal (µµµµM)

% remaining

inhibitory

activity

% Loss in

Inhibitory

activity of LC*

0 100 0

0.1 67±1 33

0. 5 42±2 58

1 82±1 18

• *The inhibitory activity of the native liver cystatin (LC) was taken as 100.

• The inhibitory activity of LC-I in the presence of ADP was assessed by its ability

to inhibit caseinolytic activity of papain as described by Kunitz.

• Concentration of LC was 1µM.