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Preparation and in vitro evaluation of radioiodinated bakuchiol as an anti tumor agent
ARTICLE IN PRESS
Applied Radiation and Isotopes 62 (2005) 389–393 www.elsevier.com/locate/apradiso
Preparation and in vitro evaluation of radioiodinated bakuchiol as an anti tumor agent Ketaki Bapata, G.J. Chintalwarb, Usha Pandeya, V.S. Thakurc, H.D. Sarmac, Grace Samuela, M.R.A. Pillaia, S. Chattopadhyayb, Meera Venkatesha, a
Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085, India b Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai 400085, India c Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085, India Received 19 March 2004; received in revised form 8 June 2004; accepted 15 July 2004
Abstract Bakuchiol, extracted from the plant Psoralea corylifolia, has been proven to have anti-tumor, cytotoxic, antimicrobial and anti-inflammatory activity. In order to study if radiolabeled bakuchiol exhibits enhanced cytotoxicity, bakuchiol was radiolabeled with 125I. In-vitro uptake studies of 125I-bakuchiol were carried out using LS-A (lymphosarcoma) and barcl-95 (radiation-induced thymic lymphoma) ascitic and solid tumor cells of murine origin. In both LS-A and barcl-95, 125I-bakuchiol showed significant uptake. Viability studies showed that the radioiodinated compound showed greater cytotoxic effect than bakuchiol. r 2004 Elsevier Ltd. All rights reserved. Keywords: Psoralea corylifolia;
125
I-bakuchiol; Anti-tumor activity; Lymphosarcoma; Radiation-induced T-cell lymphoma
1. Introduction In the past decade, therapeutic radiopharmaceuticals have been gaining attention and are currently considered to play an important role in the palliation of bone pain in patients with skeletal metastasis (Bouchet et al., 2000). The use of therapeutic radiopharmaceuticals in other malignancies, though limited at present, has also been the focus of attention in recent times. This has resulted in a variety of radiolabeled biomolecules with therapeutic potential. Generally, radioisotopes emitting a and b radiations are used for therapy. However, lowenergy Auger electrons can also be of therapeutic value, Corresponding author. Tel.: +91-22-25593676; fax: +91-
22-25505345 E-mail address: [email protected] (M. Venkatesh).
provided their energy deposition could be ensured close to the target DNA. Thus, if a biomolecule labeled with an Auger electron-emitting radionuclide (such as 125I, 117m Sn etc.) could be targeted to the nucleus of the diseased cell, it could destroy the cell and thus behave as a therapeutic agent. The use of 125I-labeled nucleotide analogs to treat various cancers have been reported (Ercan and Caglar, 2000). Here, the product is directly incorporated in DNA leading to its damage by the cascade of Auger electrons. The meroterpene phenol, bakuchiol (Fig. 1) is abundant in the seeds and leaves of the plant Psoralea corylifolia (Latha and Panikkar, 1999). Bakuchiol is widely used in Indian as well as in Chinese medicine to treat a variety of diseases and possesses anti-tumor, antibacterial, cytotoxic and anti-helmenthic properties (Latha et al., 2000). Besides possessing DNA polymerase1 inhibiting activity (Sun et al., 1998), bakuchiol is
0969-8043/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.07.007
ARTICLE IN PRESS K. Bapat et al. / Applied Radiation and Isotopes 62 (2005) 389–393
390
2.2. Tumor cells Carcinoma cells of murine origin LS-A (lymphosarcoma) (Thakur et al., 1990) and barcl-95 (radiation induced solid thymic lymphoma) (Sarma and Bhattacharjee, 1998) propagated in-vivo in Swiss mice, were obtained from colleagues working at another laboratory in our Institute. LS-A and barcl-95 tumor cells were collected from the animals bearing respective tumors.
HO Fig. 1. Structure of bakuchiol.
3. Methods reported to protect lipids and proteins against oxidative damages (Adhikari et al., 2003). In addition, it also shows hepatoprotective activity even at a low concentration (1 mg/ml) in tacrine-induced cytotoxic Hep G2 (human liver carcinoma) (Cho et al., 2001). Invitro studies with bakuchiol (20 mg/ml) against dental plaqueforming microorganism showed anti-microbial activity (Katsura et al., 2001). We envisaged that incorporation of 125I in bakuchiol might augment its cytotoxic activity. The paper describes the studies on radioiodination of bakuchiol with 125I and its cytotoxic effects in lymphoma and lymphosarcoma cells of murine origin.
2. Materials 2.1. Chemicals Bakuchiol was isolated from a P. corylifolia plant and characterized by our co-workers following reported methods (Banerji and Chintalwar, 1984; Mehta et al., 1973; Prakasa Rao et al., 1973). Elemental analysis showed %C 83.75, %H 9.43 (C18H24O) while the expected values are %C 84.32 and %H 9.44. Mono and disodium phosphate salts and sodium metabisulfite were purchased from M/s. Sarabhai Chemicals Company, India. Chloramine-T was purchased from Sigma Chemical Company, USA. Organic solvents were purchased from E. Merck. Carrier-free 125I-sodium iodide of specific activity 0.55-0.63 GBq/mg (15–17 mCi/mg) was obtained from Izotop, Budapest, Hungary. Whatman 3 mm chromatography paper was purchased from Whatman Ltd, England. Radioactivity measurements were carried out using a NaI(Tl) solid scintillation counter obtained from Electronic Corporation of India Ltd. The high-performance liquid chromatography (HPLC) system used was from Jasco, Japan (PU 1580). The system was equipped with a PU 1575 UV/Vis detector as well as with a well-type NaI(Tl) scintillation detector for measurement of radioactivity. All the solvents used for HPLC analysis were of HPLC grade, degassed and filtered before use.
3.1. Radioiodination of bakuchiol Bakuchiol was radioiodinated using chloramine-T as an oxidizing agent (Sakahara et al., 1988). In brief, to 100 mg bakuchiol in 100 ml methanol, 30 ml of 0.1 M phosphate buffer (pH 7.5) was added, followed by 3.737 MBq of Na125I. A total of 20 ml of 2 mg/ml solution of chloramine-T in 0.05 M PBS (pH 7.5) was then added to the mixture, which was then stirred for 60 s, and the reaction was terminated by addition of 20 ml of 5 mg/ml solution of sodium metabisulfite. The iodinated bakuchiol was purified from the aqueous reaction mixture by extraction into benzene. Benzene was evaporated by purging with nitrogen and 125I-bakuchiol was reconstituted in 0.1 M phosphate buffer (pH 7.5). The radiochemical purity of the purified 125I-bakuchiol was estimated by paper electrophoresis using Whatman 3 mm paper (300 V, 0.025 M phosphate buffer, pH 7.5, 60 min). Characterization of the purified product was carried out using HPLC (water (A)/methanol (B)) as the mobile phase using gradient elution (0–1 min 95% A, 1–15 min 95–5% A, 15–16 min 5% A, 16–19 min 5–95% A). The flow rate was maintained at 1 ml/min. The stability of 125I-bakuchiol was also ascertained by paper electrophoresis. 3.2. In vitro cell uptake studies In order to determine the uptake of 125I-bakuchiol in tumor cells, in-vitro experiments were carried out in LSA and barcl-95 cells of murine origin. The cells were collected from the ascitic fluid and the solid tumor, respectively. The cells were washed twice and suspended in 0.1 M phosphate buffer saline (PBS, pH 7.5). Studies were carried out using 106 cells/tube (LS-A or barcl95) at different molar concentrations of 125I-bakuchiol (1.25, 2.5, 6.25, 12.5 mM). A blank was prepared with Na 125I having a comparable count rate to the cell samples in order to determine the degree of non-specific binding. In order to estimate the uptake of 125 I-bakuchiol in normal cells, an identical experiment was carried out using normal mouse splenocytes. The cells were incubated at 371C for 30 min, at the end of
ARTICLE IN PRESS K. Bapat et al. / Applied Radiation and Isotopes 62 (2005) 389–393
which they were washed twice with cold 0.1 M PBS and the radioactivity associated with the cells was determined.
140
3.3. Cell viability studies
100
4. Results and discussion 125
The yield of I-bakuchiol was 80% as estimated by paper electrophoresis. The radiolabeled product gets preferentially extracted (495%) in benzene. This could be completely dissolved in 0.1 M phosphate buffer (pH 7.5) and its radiochemical purity was found to be 95% by paper electrophoresis (Fig. 2). Fig. 3 shows the HPLC pattern of 125I-bakuchiol. 125I-bakuchiol has a retention time of 19.2 min, which is close to the retention time for inactive bakuchiol as identified from the UV peak, while free iodide has a retention time of 2.2 min. It was observed that the radioiodinated bakuchiol retained 495% radiochemical purity even on storage for 1 month at 41C. However, at higher temperatures the product may be labile on storage as indicated by a slight drop in radiochemical purity to 94% after 4 days of storage at 371C. Significant uptake of 125I-labeled bakuchiol was observed both in LS-A and barcl-95 cells. The results of the uptake of 125I-bakuchiol by LS-A cells are presented in Table 1. Cell uptake was found to increase from 26% to 39% when the concentration of the 70 125
I-bakuchiol
60
% Radioactivity
50 40 30 20 10 125
I
0 2
4
6
8
10
12
14
16
18
20
Distance from point of spotting (cm)
Fig. 2. Paper electrophoresis pattern of purified 125I-bakuchiol.
125
I-Bakuchiol
120
80 cps
106 cells per tube (LS-A/barcl-95) were suspended in 300 ml of 0.1 M PBS (pH 7.5). 125I-bakuchiol was added to these tubes at concentrations of 1.25, 2.5, 6.25 and 12.5 mM. A similar set of experiments were carried out with inactive bakuchiol at the same concentrations. The cells were incubated at 371C for 30 min. At the end of incubation, the cells were washed twice with cold 0.1 M PBS and the supernatant was discarded. The viability of bakuchiol- (radiolabeled and unlabeled) treated cells were tested using 0.2% trypan blue dye exclusion.
391
60
125
I
40 20 0 0
200
400
600
800 1000 Time (sec)
Fig. 3. HPLC characterization of
Table 1 Uptake studies of 30 min)
1200
1400
1600
125
I-bakuchiol.
125
I-bakuchiol in lymphoma cells (37 1C,
1.25 Concentration of I-bakuchiol (mM)
2.5
6.25
12.5
125
% Cell uptakea % Blanka a
26.571 39.571 40.571 4172 2.470.5 1.670.4 1.170.06 1.270.04
(Mean7SD, n=3).
Table 2 Uptake studies of 30 min)
125
I- bakuchiol in barcl-95 cells (37 1C,
1.25 Concentration of 125 I-bakuchiol (mM) % Cell uptakea % Blanka a
2.5
6.25
12.5
17.670.5 31.670.4 26.470.5 20.270.6 0.970.1 0.770.4 1.470.1 1.170.3
(Mean7SD, n=3).
compound was increased from 1.25 to 2.5 mM. Thereafter, the cell uptake changed only marginally with the concentration. Table 2 shows the uptake of 125I-bakuchiol by barcl95 cells. In this case, however, the uptake was much less compared to that of LS-A cells. Thus, while at a concentration of 2.5 mM of 125I-bakuchiol, the barcl-95 cells showed maximum 32% uptake, the same with LS-A cells was 40%. Uptake of radiolabeled bakuchiol by normal splenocytes was found to be comparatively low (7–8%). Indeed, the extent of uptake did not change significantly (Table 3) within the concentration range 1.25, 2.5, 6.25 and 12.5, 25 mM of bakuchiol. The viability of the above tumor cell lines, in presence of both bakuchiol and 125I-bakuchiol, was studied by trypan blue dye exclusion. Treatment of LS-A cells with
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392 Table 3 Uptake of
Table 5 Viability studies in barcl-95 cells
125
I-bakuchiol in normal splenocytes
Concentration of 125 I-bakuchiol (mM)
% Uptake by splenocytesa
Concentration (mM)
% Cell viability (125I-bakuchiol)a
% Cell viability (Bakuchiol)a
1.25 2.5 6.25 12.5 25
10.570.3 7.970.1 6.570.3 5.670.1 4.770.1
2.5 25
4571.0 3772.5
7174 6375
a
%Viability of control cells was 9371.2. a (Mean7SD, n=3).
(Mean7SD, n=2).
Table 4 Viability studies in LS-A (lymphosarcoma) cells Concentration (mM)
% Viability (125I-bakuchiol)a
% Viability (bakuchiol)a
1.25 2.5 6.25 12.5
7470.6 4472.5 2370.1 1472.5
83.372.4 8074.9 6874.2 5774.2
% Viability of control cells was 9571.4. a (Mean7SD, n=3). 125
I-bakuchiol resulted in marked decrease in their viability. The effect was concentration dependent and 125 I-bakuchiol was very effective even at very low concentrations. Although the results with unlabeled bakuchiol showed similar trends (Table 4), the effect was significantly higher with 125I-bakuchiol. With the same concentration (2.5 mM) of bakuchiol and its labeled counterpart, the viability of the LS-A cells were 8074.9% and 4472.5% respectively. The results of the viability studies of barcl-95 cells in presence of 125I-bakuchiol are presented in Table 5. In this case also, treatment with 125I-bakuchiol led to a significant reduction in the cell viability. For example, at the same concentration (2.5 mM) of bakuchiol and its labeled counterpart, viability of barcl-95 cells were 7174% and 4571.0%. As in the case of LS-A cells, the radiolabeling of bakuchiol augmented its cell-killing activity. The viability of the normal splenocytes was unaffected by 125I-bakuchiol. A variety of radiopharmaceuticals have been introduced for the internal therapy of malignant and inflammatory lesions in nuclear medicine. The Auger and conversion electrons have low energies and low range (up to 10 nm). These, if targeted to reach the nucleus of cells, could kill the cells and yet leave the nontargeted area unharmed due to low energy and range. Thus, radiopharmaceuticals containing an Auger electron-emitting isotope have been proposed for therapeutic application (Ercan and Caglar, 2000). In order to determine whether bakuchiol labeled with 125I exhibits enhanced cytotoxicity, 125I-bakuchiol was prepared and
evaluated in murine lymphosarcoma (LS-A) cells and lymphoma (barcl-95) solid tumor cells. For this, the viability of the above tumor cells after treatment with the 125I-labeled and unlabeled bakuchiol was compared. The results showed that the cytotoxicity of bakuchiol is enhanced upon radioiodination without affecting its mode of action. Some researchers have earlier studied the cytotoxicity of bakuchiol using the L929 cell line (Kubo et al., 1989; Iwamura et al., 1989) and have established the structure–activity correlation. The alkyl group was found to be obligatory for the activity, which was marginally influenced by the olefinic functionality. Based on electron microscopic observation and hemolytic activity, its mechanism of action has been proposed to be due to an injury of cell membrane. In comparison, the present study revealed for the first time that radiolabeling of bakuchiol with the Auger-emitting isotope 125I could augment its toxicity to the tumor cells appreciably. The fact that the radiolabeled product clearly shows increased cytotoxic effect, suggests that the molecule may be entering the cell, which opens new areas to be explored such as the actual mode of action in normal splenocytes and cancer cells. As the anti-tumor activity is not due to receptor specificity, 125I-bakuchiol could be studied with various tumor cell lines of human origin.
5. Conclusion Bakuchiol could be radioiodinated easily due to the presence of a phenolic group at 80% radiolabeling yield and purified by a simple procedure to obtain 495% pure radioiodinated bakuchiol. LS-A and barcl95 cells exhibited high uptake of the radiolabeled bakuchiol. Radiolabeling has enhanced the cytotoxic nature of bakuchiol significantly as evident from the viability studies. As 125I-bakuchiol did not hamper the survival of mouse splenocytes while affecting the tumor cell viability, it has the potential to be effective as a cytotoxic agent in other tumor cells. Therefore, biodistribution studies in murine tumor models and in vitro uptake studies in various tumor cell lines of human origin could be studied to test the efficacy of 125 I-bakuchiol as a therapeutic agent.
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References Adhikari, S., Joshi, R., Patro, B.S., Ghanty, T.K., Chintalwar, G.J., Sharma, A., Chattopadhyay, S., Mukherjee, T., 2003. Anti-oxidant activity of bakuchiol: experimental evidences and theoretical treatments on the possible involvement of the terpenoid chain. Chem. Res. Toxicol. 16 (9), 1062–1069. Banerji, A., Chintalwar, G.J., 1984. Some aspects of monoterpenens biosynthesis. Proc. Indian Acad. Sci. (Chem. Sci.) 93 (7), 1171–1178. Bouchet, L.G., Bolch, W.E., Goddu, S.M., Howell, R.W., Rao, D.V., 2000. Considerations in the selection of radiopharmaceuticals for palliation of bone pain from metastatic osseous lesions. J. Nucl. Med. 41 (4), 682–687. Cho, H., Jun, J.Y., Song, E.K., Kang, K.H., Baek, H.Y., Ko, Y.S., Kim, Y.C., 2001. Bakuchiol: a hepatoprotective compound of psoralea corylifolia on tacrine induced cytotoxicity in HepG2 cells. Planta Med. 67 (8), 750–751. Ercan, M.T., Caglar, M., 2000. Therapeutic radiopharmaceuticals. In: Current Pharmaceutical Design. 6 (11), 1085–1121. Iwamura, J., Dohi, T., Tanaka, H., Odani, T., Kubo, M., 1989. Cytotoxicity of Corylifoliae fructus II. Cytotoxicity of bakuchiol and the analogues. Yakuga. Zasshi 109 (12), 962–965. Katsura, H., Tsutikiyama, R., Suzuki, A., Kobayashi, M., 2001. In-vitro anti microbial activities of bakuchiol against oral microorganisms. Anti Microbial Agents Chemother. 45 (11), 3009–3013. Kubo, M., Dohi, T., Odani, T., Tanaka, H., Iwamura, J., 1989. Cytotoxicity of Corylifoliae fructus I. Isolation of the effective compound and the cytotoxicity. Yakuga. Zasshi 109 (12), 926–931.
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Latha, P.G., Evans, D.A., Panikkar, K.R., Jayawardhanan, K.K., 2000. Immunomodulatory and anti tumor properties of Psoralea corylifolia seeds. Fitoterpia 71 (3), 223–231. Latha, P.G., Panikkar, K.R., 1999. Inhibition of chemical carcinogenesis by Psoralea corylifolia seeds. J. Ethnopharmacol. 68 (1–3), 295–298. Mehta, G., Nayak, U.R., Dev, S., 1973. Meroterpenoids-I; Psoralea corylifolia Linn. –1. Bakuchiol, a novel monoterpene phenol. Tetrahedron 29 (8), 1119–1125. Prakasa Rao, A.S.C., Bhalla, V., Nayak, U.R., Dev, S., 1973. Meroterpenoids-II; Psoralea corylifolia Linn.-2. Absolute configuration of (+) bakuchiol. Tetrahedron 29 (8), 1127–1130. Sakahara, H., Endo, K., Koizumi, M., Nakashima, T., Kunimatsu, M., Watanabe, Y., Kawamura, Y., Nakamura, T., Tanaka, H., Kotoura, Y., Yamamuro, Y., Hosoi, S., Toyama, S., Torizuka, K., 1988. Relationship between in vitro binding activity and in vivo tumor accumulation of radiolabelled monoclonal antibodies. J. Nucl. Med. 29 (2), 235–240. Sarma, H.D., Bhattacharjee, D., 1998. Radiation sensitivity to a new transplantable tumor ‘barcl-95’ derived from radiation induced thymic lymphoma. In: Sharan R.N. (Ed.), Trends in Radiation and Cancer Biology, Vol. 29, pp. 100–103. Sun, N.J., Woo, S.H., Cassady, J.M., Snapka, R.M., 1998. DNA Polymerase and topoisomerase II inhibitors from Psoralea corylifolia. J. Nat. Prod. 61 (3), 362–366. Thakur, V.S., Seshadri, M., Poduwal, T.B., Shah, D.H., Sundaram, K., 1990. Allosensitisation induced suppression of various murine tumors: role of non H-2 antigens in antitumor immunity. Indian J. Exp. Biol. 28, 706–710.
Applied Radiation and Isotopes 62 (2005) 389–393 www.elsevier.com/locate/apradiso
Preparation and in vitro evaluation of radioiodinated bakuchiol as an anti tumor agent Ketaki Bapata, G.J. Chintalwarb, Usha Pandeya, V.S. Thakurc, H.D. Sarmac, Grace Samuela, M.R.A. Pillaia, S. Chattopadhyayb, Meera Venkatesha, a
Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085, India b Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai 400085, India c Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085, India Received 19 March 2004; received in revised form 8 June 2004; accepted 15 July 2004
Abstract Bakuchiol, extracted from the plant Psoralea corylifolia, has been proven to have anti-tumor, cytotoxic, antimicrobial and anti-inflammatory activity. In order to study if radiolabeled bakuchiol exhibits enhanced cytotoxicity, bakuchiol was radiolabeled with 125I. In-vitro uptake studies of 125I-bakuchiol were carried out using LS-A (lymphosarcoma) and barcl-95 (radiation-induced thymic lymphoma) ascitic and solid tumor cells of murine origin. In both LS-A and barcl-95, 125I-bakuchiol showed significant uptake. Viability studies showed that the radioiodinated compound showed greater cytotoxic effect than bakuchiol. r 2004 Elsevier Ltd. All rights reserved. Keywords: Psoralea corylifolia;
125
I-bakuchiol; Anti-tumor activity; Lymphosarcoma; Radiation-induced T-cell lymphoma
1. Introduction In the past decade, therapeutic radiopharmaceuticals have been gaining attention and are currently considered to play an important role in the palliation of bone pain in patients with skeletal metastasis (Bouchet et al., 2000). The use of therapeutic radiopharmaceuticals in other malignancies, though limited at present, has also been the focus of attention in recent times. This has resulted in a variety of radiolabeled biomolecules with therapeutic potential. Generally, radioisotopes emitting a and b radiations are used for therapy. However, lowenergy Auger electrons can also be of therapeutic value, Corresponding author. Tel.: +91-22-25593676; fax: +91-
22-25505345 E-mail address: [email protected] (M. Venkatesh).
provided their energy deposition could be ensured close to the target DNA. Thus, if a biomolecule labeled with an Auger electron-emitting radionuclide (such as 125I, 117m Sn etc.) could be targeted to the nucleus of the diseased cell, it could destroy the cell and thus behave as a therapeutic agent. The use of 125I-labeled nucleotide analogs to treat various cancers have been reported (Ercan and Caglar, 2000). Here, the product is directly incorporated in DNA leading to its damage by the cascade of Auger electrons. The meroterpene phenol, bakuchiol (Fig. 1) is abundant in the seeds and leaves of the plant Psoralea corylifolia (Latha and Panikkar, 1999). Bakuchiol is widely used in Indian as well as in Chinese medicine to treat a variety of diseases and possesses anti-tumor, antibacterial, cytotoxic and anti-helmenthic properties (Latha et al., 2000). Besides possessing DNA polymerase1 inhibiting activity (Sun et al., 1998), bakuchiol is
0969-8043/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.07.007
ARTICLE IN PRESS K. Bapat et al. / Applied Radiation and Isotopes 62 (2005) 389–393
390
2.2. Tumor cells Carcinoma cells of murine origin LS-A (lymphosarcoma) (Thakur et al., 1990) and barcl-95 (radiation induced solid thymic lymphoma) (Sarma and Bhattacharjee, 1998) propagated in-vivo in Swiss mice, were obtained from colleagues working at another laboratory in our Institute. LS-A and barcl-95 tumor cells were collected from the animals bearing respective tumors.
HO Fig. 1. Structure of bakuchiol.
3. Methods reported to protect lipids and proteins against oxidative damages (Adhikari et al., 2003). In addition, it also shows hepatoprotective activity even at a low concentration (1 mg/ml) in tacrine-induced cytotoxic Hep G2 (human liver carcinoma) (Cho et al., 2001). Invitro studies with bakuchiol (20 mg/ml) against dental plaqueforming microorganism showed anti-microbial activity (Katsura et al., 2001). We envisaged that incorporation of 125I in bakuchiol might augment its cytotoxic activity. The paper describes the studies on radioiodination of bakuchiol with 125I and its cytotoxic effects in lymphoma and lymphosarcoma cells of murine origin.
2. Materials 2.1. Chemicals Bakuchiol was isolated from a P. corylifolia plant and characterized by our co-workers following reported methods (Banerji and Chintalwar, 1984; Mehta et al., 1973; Prakasa Rao et al., 1973). Elemental analysis showed %C 83.75, %H 9.43 (C18H24O) while the expected values are %C 84.32 and %H 9.44. Mono and disodium phosphate salts and sodium metabisulfite were purchased from M/s. Sarabhai Chemicals Company, India. Chloramine-T was purchased from Sigma Chemical Company, USA. Organic solvents were purchased from E. Merck. Carrier-free 125I-sodium iodide of specific activity 0.55-0.63 GBq/mg (15–17 mCi/mg) was obtained from Izotop, Budapest, Hungary. Whatman 3 mm chromatography paper was purchased from Whatman Ltd, England. Radioactivity measurements were carried out using a NaI(Tl) solid scintillation counter obtained from Electronic Corporation of India Ltd. The high-performance liquid chromatography (HPLC) system used was from Jasco, Japan (PU 1580). The system was equipped with a PU 1575 UV/Vis detector as well as with a well-type NaI(Tl) scintillation detector for measurement of radioactivity. All the solvents used for HPLC analysis were of HPLC grade, degassed and filtered before use.
3.1. Radioiodination of bakuchiol Bakuchiol was radioiodinated using chloramine-T as an oxidizing agent (Sakahara et al., 1988). In brief, to 100 mg bakuchiol in 100 ml methanol, 30 ml of 0.1 M phosphate buffer (pH 7.5) was added, followed by 3.737 MBq of Na125I. A total of 20 ml of 2 mg/ml solution of chloramine-T in 0.05 M PBS (pH 7.5) was then added to the mixture, which was then stirred for 60 s, and the reaction was terminated by addition of 20 ml of 5 mg/ml solution of sodium metabisulfite. The iodinated bakuchiol was purified from the aqueous reaction mixture by extraction into benzene. Benzene was evaporated by purging with nitrogen and 125I-bakuchiol was reconstituted in 0.1 M phosphate buffer (pH 7.5). The radiochemical purity of the purified 125I-bakuchiol was estimated by paper electrophoresis using Whatman 3 mm paper (300 V, 0.025 M phosphate buffer, pH 7.5, 60 min). Characterization of the purified product was carried out using HPLC (water (A)/methanol (B)) as the mobile phase using gradient elution (0–1 min 95% A, 1–15 min 95–5% A, 15–16 min 5% A, 16–19 min 5–95% A). The flow rate was maintained at 1 ml/min. The stability of 125I-bakuchiol was also ascertained by paper electrophoresis. 3.2. In vitro cell uptake studies In order to determine the uptake of 125I-bakuchiol in tumor cells, in-vitro experiments were carried out in LSA and barcl-95 cells of murine origin. The cells were collected from the ascitic fluid and the solid tumor, respectively. The cells were washed twice and suspended in 0.1 M phosphate buffer saline (PBS, pH 7.5). Studies were carried out using 106 cells/tube (LS-A or barcl95) at different molar concentrations of 125I-bakuchiol (1.25, 2.5, 6.25, 12.5 mM). A blank was prepared with Na 125I having a comparable count rate to the cell samples in order to determine the degree of non-specific binding. In order to estimate the uptake of 125 I-bakuchiol in normal cells, an identical experiment was carried out using normal mouse splenocytes. The cells were incubated at 371C for 30 min, at the end of
ARTICLE IN PRESS K. Bapat et al. / Applied Radiation and Isotopes 62 (2005) 389–393
which they were washed twice with cold 0.1 M PBS and the radioactivity associated with the cells was determined.
140
3.3. Cell viability studies
100
4. Results and discussion 125
The yield of I-bakuchiol was 80% as estimated by paper electrophoresis. The radiolabeled product gets preferentially extracted (495%) in benzene. This could be completely dissolved in 0.1 M phosphate buffer (pH 7.5) and its radiochemical purity was found to be 95% by paper electrophoresis (Fig. 2). Fig. 3 shows the HPLC pattern of 125I-bakuchiol. 125I-bakuchiol has a retention time of 19.2 min, which is close to the retention time for inactive bakuchiol as identified from the UV peak, while free iodide has a retention time of 2.2 min. It was observed that the radioiodinated bakuchiol retained 495% radiochemical purity even on storage for 1 month at 41C. However, at higher temperatures the product may be labile on storage as indicated by a slight drop in radiochemical purity to 94% after 4 days of storage at 371C. Significant uptake of 125I-labeled bakuchiol was observed both in LS-A and barcl-95 cells. The results of the uptake of 125I-bakuchiol by LS-A cells are presented in Table 1. Cell uptake was found to increase from 26% to 39% when the concentration of the 70 125
I-bakuchiol
60
% Radioactivity
50 40 30 20 10 125
I
0 2
4
6
8
10
12
14
16
18
20
Distance from point of spotting (cm)
Fig. 2. Paper electrophoresis pattern of purified 125I-bakuchiol.
125
I-Bakuchiol
120
80 cps
106 cells per tube (LS-A/barcl-95) were suspended in 300 ml of 0.1 M PBS (pH 7.5). 125I-bakuchiol was added to these tubes at concentrations of 1.25, 2.5, 6.25 and 12.5 mM. A similar set of experiments were carried out with inactive bakuchiol at the same concentrations. The cells were incubated at 371C for 30 min. At the end of incubation, the cells were washed twice with cold 0.1 M PBS and the supernatant was discarded. The viability of bakuchiol- (radiolabeled and unlabeled) treated cells were tested using 0.2% trypan blue dye exclusion.
391
60
125
I
40 20 0 0
200
400
600
800 1000 Time (sec)
Fig. 3. HPLC characterization of
Table 1 Uptake studies of 30 min)
1200
1400
1600
125
I-bakuchiol.
125
I-bakuchiol in lymphoma cells (37 1C,
1.25 Concentration of I-bakuchiol (mM)
2.5
6.25
12.5
125
% Cell uptakea % Blanka a
26.571 39.571 40.571 4172 2.470.5 1.670.4 1.170.06 1.270.04
(Mean7SD, n=3).
Table 2 Uptake studies of 30 min)
125
I- bakuchiol in barcl-95 cells (37 1C,
1.25 Concentration of 125 I-bakuchiol (mM) % Cell uptakea % Blanka a
2.5
6.25
12.5
17.670.5 31.670.4 26.470.5 20.270.6 0.970.1 0.770.4 1.470.1 1.170.3
(Mean7SD, n=3).
compound was increased from 1.25 to 2.5 mM. Thereafter, the cell uptake changed only marginally with the concentration. Table 2 shows the uptake of 125I-bakuchiol by barcl95 cells. In this case, however, the uptake was much less compared to that of LS-A cells. Thus, while at a concentration of 2.5 mM of 125I-bakuchiol, the barcl-95 cells showed maximum 32% uptake, the same with LS-A cells was 40%. Uptake of radiolabeled bakuchiol by normal splenocytes was found to be comparatively low (7–8%). Indeed, the extent of uptake did not change significantly (Table 3) within the concentration range 1.25, 2.5, 6.25 and 12.5, 25 mM of bakuchiol. The viability of the above tumor cell lines, in presence of both bakuchiol and 125I-bakuchiol, was studied by trypan blue dye exclusion. Treatment of LS-A cells with
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392 Table 3 Uptake of
Table 5 Viability studies in barcl-95 cells
125
I-bakuchiol in normal splenocytes
Concentration of 125 I-bakuchiol (mM)
% Uptake by splenocytesa
Concentration (mM)
% Cell viability (125I-bakuchiol)a
% Cell viability (Bakuchiol)a
1.25 2.5 6.25 12.5 25
10.570.3 7.970.1 6.570.3 5.670.1 4.770.1
2.5 25
4571.0 3772.5
7174 6375
a
%Viability of control cells was 9371.2. a (Mean7SD, n=3).
(Mean7SD, n=2).
Table 4 Viability studies in LS-A (lymphosarcoma) cells Concentration (mM)
% Viability (125I-bakuchiol)a
% Viability (bakuchiol)a
1.25 2.5 6.25 12.5
7470.6 4472.5 2370.1 1472.5
83.372.4 8074.9 6874.2 5774.2
% Viability of control cells was 9571.4. a (Mean7SD, n=3). 125
I-bakuchiol resulted in marked decrease in their viability. The effect was concentration dependent and 125 I-bakuchiol was very effective even at very low concentrations. Although the results with unlabeled bakuchiol showed similar trends (Table 4), the effect was significantly higher with 125I-bakuchiol. With the same concentration (2.5 mM) of bakuchiol and its labeled counterpart, the viability of the LS-A cells were 8074.9% and 4472.5% respectively. The results of the viability studies of barcl-95 cells in presence of 125I-bakuchiol are presented in Table 5. In this case also, treatment with 125I-bakuchiol led to a significant reduction in the cell viability. For example, at the same concentration (2.5 mM) of bakuchiol and its labeled counterpart, viability of barcl-95 cells were 7174% and 4571.0%. As in the case of LS-A cells, the radiolabeling of bakuchiol augmented its cell-killing activity. The viability of the normal splenocytes was unaffected by 125I-bakuchiol. A variety of radiopharmaceuticals have been introduced for the internal therapy of malignant and inflammatory lesions in nuclear medicine. The Auger and conversion electrons have low energies and low range (up to 10 nm). These, if targeted to reach the nucleus of cells, could kill the cells and yet leave the nontargeted area unharmed due to low energy and range. Thus, radiopharmaceuticals containing an Auger electron-emitting isotope have been proposed for therapeutic application (Ercan and Caglar, 2000). In order to determine whether bakuchiol labeled with 125I exhibits enhanced cytotoxicity, 125I-bakuchiol was prepared and
evaluated in murine lymphosarcoma (LS-A) cells and lymphoma (barcl-95) solid tumor cells. For this, the viability of the above tumor cells after treatment with the 125I-labeled and unlabeled bakuchiol was compared. The results showed that the cytotoxicity of bakuchiol is enhanced upon radioiodination without affecting its mode of action. Some researchers have earlier studied the cytotoxicity of bakuchiol using the L929 cell line (Kubo et al., 1989; Iwamura et al., 1989) and have established the structure–activity correlation. The alkyl group was found to be obligatory for the activity, which was marginally influenced by the olefinic functionality. Based on electron microscopic observation and hemolytic activity, its mechanism of action has been proposed to be due to an injury of cell membrane. In comparison, the present study revealed for the first time that radiolabeling of bakuchiol with the Auger-emitting isotope 125I could augment its toxicity to the tumor cells appreciably. The fact that the radiolabeled product clearly shows increased cytotoxic effect, suggests that the molecule may be entering the cell, which opens new areas to be explored such as the actual mode of action in normal splenocytes and cancer cells. As the anti-tumor activity is not due to receptor specificity, 125I-bakuchiol could be studied with various tumor cell lines of human origin.
5. Conclusion Bakuchiol could be radioiodinated easily due to the presence of a phenolic group at 80% radiolabeling yield and purified by a simple procedure to obtain 495% pure radioiodinated bakuchiol. LS-A and barcl95 cells exhibited high uptake of the radiolabeled bakuchiol. Radiolabeling has enhanced the cytotoxic nature of bakuchiol significantly as evident from the viability studies. As 125I-bakuchiol did not hamper the survival of mouse splenocytes while affecting the tumor cell viability, it has the potential to be effective as a cytotoxic agent in other tumor cells. Therefore, biodistribution studies in murine tumor models and in vitro uptake studies in various tumor cell lines of human origin could be studied to test the efficacy of 125 I-bakuchiol as a therapeutic agent.
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