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Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract on U937 and K562 cells
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Toxicon 48 (2006) 388–400 www.elsevier.com/locate/toxicon
Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract on U937 and K562 cells B. Giria, A. Gomesb, A. Debnathb, A. Sahaa, A.K. Biswasa, S.C. Dasguptaa, A. Gomesa, a
Laboratory of Toxinology and Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata 700 009, India b New Drug Development Division, Indian Institute of Chemical Biology, 4, Raja Subodh Mallik Road, Kolkata 700 032, India Received 20 February 2006; received in revised form 20 June 2006; accepted 22 June 2006 Available online 29 June 2006
Abstract The antiproliferative, cytotoxic and apoptogenic activities of Bufo melanostictus (Indian common toad) skin extract (TSE) on U937 and K562 leukemic cell line has been investigated. TSE significantly (Po0.001) reduced the time-dependent cell proliferation and decreased MTT values in U937 and K562 cells. TSE (IC50 doses) suppressed the proliferating cell nuclear antigen expression in both the cells. It was demonstrated that, TSE (IC50 doses) primarily arrested the U937 and K562 cells at G1 phase of the cell cycle. Confocal microscopy showed the altered fragmented nuclei and apoptotic bodies formation in TSE (IC50 doses) treated U937 and K562 cells. Membrane blebbing, cell surface shrinkage and perforation were observed through scanning electron microscope. TSE-induced DNA fragmentation in U937 and K562 cells was reflected in single-cell gel electrophoresis. TSE significantly (Po0.001) increase the length–width ratio of DNA mass as compared to control in comet assay. The flow cytometric analysis of annexin-V binding to the cancer cells further supported the apoptotogenic activity of TSE. The effect of TSE on normal human peripheral blood mononuclear cells viability and cytotoxicity was studied in culture and found to be less cytotoxic than on the U937 and K562 cells. The findings from the present study suggested that TSE might possess potent antineoplastic agent having antiproliferative, cytotoxic and apoptogenic activity against U937 and K562 myeloid leukemic cells. r 2006 Elsevier Ltd. All rights reserved. Keywords: Toad skin; Skin extract; Anticancer activity; Cytotoxic agent; U937 cell; K562 cell
1. Introduction Cancer is a complex multifactorial disease of the cell. Cancer development and progression are Corresponding author. Tel.: +91 33 2244 4755; fax: +91 33 2351 9755. E-mail address: [email protected] (A. Gomes).
0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.06.011
dependent on cellular accumulation of various genetic and epigenetic events (Blagosklonny, 2005). Radiotherapy, chemotherapy and surgical measure are the key tools for cancer treatment. In the hematopoetic malignancies, chemotherapeutic approaches are widely applied in practice. Nowadays, several therapeutic approaches have been taken to overcome the complexities of different
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cancers. Drug discovery against cancer is ventured throughout the world especially from the natural products. It is evident to note that amphibians like frog and toad possess different bioactive substances in their skin (Duellman and Trueb, 1986). Amphibian skin is a morphologically, biochemically and physiologically complex organ, which fulfils a wide range of functions necessary for the amphibian survival, including respiration, water regulation, antipredator, antimicrobial defense, excretion, temperature control, etc., (Clarke, 1997). Toads, particularly members of the genus Bufo, are identified as a particularly convenient and useful source of granular gland secretions, which commonly contain biogenic amines, bufodienolides, alkaloids and steroids, peptides and proteins (Cei et al., 1967; Clarke, 1997; Daly et al., 2004; Maciel et al., 2003; Steyn and Van Heerden, 1998). In the Chinese traditional medicine, amphibian’s skin extract had been used for the alleviation of human sufferings (Clarke, 1997; Ko et al., 2005). Chan Sue, the Chinese toad (Bufo bufo gargarizans) skin extract preparation was used in the treatment of various diseases, like cancer, arrhythmia and other heart diseases. (Datta and Dasgupta, 2002; Bhuiyan et al., 2003; Nogawa et al., 2001). A number of research is going on in other parts of the world regarding the anticancer activities of different toad and frog skin components; but few scientific information are available on the Indian toad and frog skin bioactive compound. Earlier investigation from this laboratory revealed that Indian toad (Bufo melanostictus) skin extract is pharmacologically potent having immunomodulatory and antineoplastic activity on Ehrlich ascites carcinoma bearing mice (Das et al., 1998a; Das et al., 1998b). The present communication is an approach to study the antiproliferative, cytotoxic and apoptogenic activities of Indian toad (B. melanostictus) skin extract on U937 and K562 cells. 2. Materials and methods
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mesylate (Natco, India), low melting point agarose (Promega, USA), methanol (Spectrochem, India), MTT (Sigma, USA), normal melting point agarose (Promega, USA), osmium tetraoxide (Sigma, USA) Penicillin–streptomycin (Biowest, Germany) proliferating cell nuclear antigen (PCNA) antibody (Santa Cruz Biotechnology, Inc., USA) phytohemagglutinin (Sigma-Aldrich, USA), polylysine (Sigma, USA), propidium iodide (Sigma, USA), RPMI (GIBCO BRL, USA), sodium cacodylate (SRL, India), Triton X-100 (SRL, India), trypan blue (SRL, India). 2.2. Collection and preparation of toad skin extract (TSE) Adult toads (B. melanostictus, Schneider) of both sexes (80710 g) were collected commercially from Ms. Reeta Ghosh & Company, Calcutta, India. Skin was collected from freshly pithed toad, washed in distilled water, weighed and immersed in 99.5% methanol at room temperature for 30–40 days. The supernatant was collected and filtered. It was evaporated to dryness by rotary evaporator and the extract was kept at room temperature in a desiccator. The yield of the extract was 1.370.46% of the skin and expressed in terms of dry weight. The extract (TSE) was suspended in RPMI medium for the experiments. 2.3. Human myeloid leukemic cells and culture U937 and K562 cell lines were procured from the National Facility for Animal Tissue and Cell culture, Pune, India. Cell line was used after 17 passages. U937 and K562 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (heat inactivated), penicillin (100 units/ml), streptomycin (100 mg/ml) and gentamicin (100 mg/ml). Cell lines were maintained in a humidified incubator with 5% CO2 and 95% air at 37 1C. It was subcultured weekly at an initial density of 1 106 cells/ ml and maintained.
2.1. Chemicals used Annexin-V antibody (Sigma, USA), Ara-C (Sigma, USA), Dimethyl sulfoxide (DMSO) (SRL, India), EDTA (Sigma, USA), ethidium bromide (Sigma, USA), fetal bovine serum (GIBCO, BRL, USA), gentamicin (Nicholas, India), glutaraldehyde (Merck, India), HEPES (Sigma, USA), heparin (Sigma, USA), histopaque (Sigma, USA), imatinib
2.4. Normal human peripheral blood mononuclear cell culture and its viability and cytotoxicity studies After informed consent, 5 ml of blood was drawn (venipuncture) aseptically from healthy human volunteers (25–30 years of age) and transferred into sterile tubes containing 10 U/ml heparin inside a laminar flow hood. Heparinized blood was diluted
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with equal volume of normal saline. About 10 ml of diluted blood was carefully layered on 3 ml of histopaque. It was then centrifuged at 1500 rpm for 20 min at room temperature. Peripheral blood mononuclear cells (PBMNC) were collected from histopaque–plasma interface and washed twice with normal saline. The cell pellet was resuspended in culture medium (as mentioned in Section 2.3.) with density of 1 106 cells/ml and cultured in a CO2 incubator with 95% O2 and 5% CO2 at 37 1C for 24 h in presence and absence of TSE (100 mg/ml) and standard anticancer drugs imatinib mesylate (100 mg/ml) and Ara-C (100 mg/ml). PBMNCs were stimulated with 2.5 mg/ml of phytohemoagglutinin (Kondala et al., 2005). Cell viability was determined using Trypan blue (0.4%) dye exclusion procedure and viable cells were counted in a phase contrast microscope (Olympus CK40) and percentage (%) inhibition of viable cells was calculated as compared with control. The extent of cytotoxicity was analyzed and expressed as % inhibition of OD values in MTT assay (as mentioned in Section 2.7.) (Kawada et al., 2002). 2.5. Determination of IC50 The 50% of inhibition concentration (IC50) values of TSE in U937 and K562 cells were determined using different concentrations (25, 50, 75, 100, 125, 150 mg/ml) of the extract at 24 h. The respective viable cells were counted in the phase contrast microscope using Trypan blue (Agrawal et al., 1989). The respective concentration of extract was converted to their respective log dose. The percentage of cell death was converted to Probit value in respective dose concerned. The graph was plotted with the different log dose of the extract against the respective Probit value using Origin 6.0 Professional software. The IC50 value was noted. The above mentioned procedure were repeated four times and the confidence intervals of IC50 values were calculated.
was incubated in a humidified 5% CO2 incubator for 24, 48 and 72 h at 37 1C. The number of viable cells was determined by the Trypan blue dye exclusion test and the percentage inhibition of cell growth was calculated as compared with control (Banks-Schlegel and Quintero, 1986; Agrawal et al., 1989). 2.7. MTT assay MTT assay was done according to Kawada et al., (2002). One hundred microliters of myeloid leukemic cells (U937 and K562) suspension in RPMI 1640 and supplemented with 10% foetal calf serum containing 1 105 cells were added to each well of a 96-well microtiter plate separately. Cancer cells were cultured in presence and absence of TSE (100 mg/ml). Plates were incubated at 37 1C in a humidified 5% CO2 incubator for 24 h. Twenty microliters of MTT (5 mg/ml) was then added in each well and allowed to incubate at 37 1C in 5% CO2 incubator for 3 h. 100 ml DMSO was added to each well to dissolve the formazan crystal formed. The OD was recorded at 570 nm with microplate reader (Merck-MIOS Mini, Model No. 309). Growth inhibitory rate (%) was expressed as ¼ [1(OD of treated/OD of control)] 100. 2.8. Proliferating cell nuclear antigen (PCNA) expression After 24 h of TSE treatment (IC50 doses), U937 and K562 cells were mechanically and enzymatically dissociated to obtain bare nuclei, treated with RNAase, then stained with propidium iodide. The nuclei suspensions were acquired and analyzed on TM Becton Dickinson (BD LSR three laser Flow Cytometer, using 488/UV/HeNe 488 nm Argon ion/ 325 nm HeCd/633 nm laser) and internal signals from the blue photomultiplier transducer. A total events of 10,000 cells were collected for each sample. PCNA expression was done using antihuman rabbit PCNA antibody and expressed as percentage positivity (Giordano et al., 1991).
2.6. Cell growth inhibition study 2.9. Cell cycle analysis To observe cell growth inhibitory effects, 100 ml of myeloid leukemic cell (U937 and K562) suspension containing 1 106 cells/ml in culture medium in each well was added in 96-well microtitre plate separately. The TSE was dissolved in sterile RPMI medium and cancer cells were cultured in presence and absence of TSE (100 mg/ml). The culture plate
For the analysis of cell cycle phase distribution of DNA, respective control, standard and TSE-treated cancer cells (1 106 each) were washed with PBS after 24 h of TSE treatment (IC50 doses) and fixed in 70% ethanol and stained with propidium iodide (100 mg/ml in 0.1% sodium citrate containing 0.1%
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Triton X-100) after RNAase (10 mg/ml) treatment (Lim et al., 1999). The cell cycle distribution was determined by flow cytometry (Becton Dickinson, TM BD LSR three-laser Flow Cytometer) using CellQuest software. Fluorescence detector equipped with 488 nm Argon laser light source and 623 nm band pass filter. Total of 10,000 cells were acquired for analysis. 2.10. Annexin-V binding study U937 and K562 cells (1 106) were cultured in presence and absence of TSE (IC50 doses) for 24 h. The cells were then washed with phosphate buffer saline and centrifuged at 1500 rpm at 4 1C. The cell pellet was washed twice with 1X Annexin–Hepes buffer and resuspended in 100 ml of this buffer. Annexin-V antibody (final concentration of 0.2 mg/ml) was added to this cell suspension and incubated for 30 min in ice in the dark room. Then it was washed again with 1X Annexin–Hepes buffer and fixed with 1% paraformaldehyde at 4 1C. The cells were analyzed within 3–4 h by flow cytometry (Becton Dickinson, FACS Calibur) with Cell Quest software. Flow cytometer was set for collecting data of 10,000 cells in each group (Vermes et al., 1995). 2.11. Confocal microscopy Cancer cells (1 105) were harvested and centrifuged at 4000 rpm for 4 min and washed with PBS after 24 h of TSE (IC50 doses) treatment. The pellet was then suspended in 100 ml of PBS. Ten microliters of cell suspension was spotted on polylysinecoated coverslip and incubated at 4 1C for 30 min. It was then washed thrice with PBS. Methanol/ acetone (1:1) mixture was added on it and kept for 5 min. It was again washed with PBS. Ten microliters of ethidium bromide (100 mg/ml) containing 10% glycerol was taken on the fixed cells smear of the coverslip. It was then covered with a prepolylysine-coated coverslip. The prepared cell sandwich was kept in a moist petri dish overnight before analysis. It was then analyzed in a laser scanning confocal microsope (Leica Model SP2) (Guan et al., 2004). 2.12. Scanning electron microscopy U937 and K562 cells (1 105) were cultured in presence and absence of TSE (IC50 doses) for 48 h. The cells were then washed twice with phosphate
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buffer saline (pH 7.2) and centrifuged at 1500 rpm at 4 1C. It was fixed with 2.5% glutaraldehyde at 4 1C for 3 h in dark. The samples were again washed with PBS and post-fixed in 1% OsO4 in a cacodylate buffer for 1 h. Finally, cells were dehydrated in ascending grade of ethanol and embedded in polylysine-coated thick cover glass. The prepared cells on the cover glass were then gold coated and analyzed at 15 KV accelerating voltage by Leica (Model S440) scanning electron microscope (Nottola et al., 2005). 2.13. Single-cell gel electrophoresis (alkaline comet assay) Comet assay of U937 and K562 cells was performed under alkaline condition following method of Singh et al., (1988) with minor modifications. U937 and K562 cells (1 105) were cultured in presence and absence of TSE (IC50 doses) for 48 h. The cells were then washed twice with cold PBS by centrifuging at 1500 rpm for 5 min in cold centrifuge. Concentration (1–2 104/10 ml) of different cell groups was standardized by changing the dilution of cell suspension. Microscope slides were covered with 400 ml of 0.75% normal melting point agarose in PBS pre-warmed to 50 1C. A cover glass was placed over the agarose solution, and the agarose allowed to solidify. The coverglass was then removed, and 85 ml of cell–agarose suspension (10 ml of cell suspension containing about 104 cells was mixed with 75 ml of 0.5% low melting point agarose, in PBS) was placed over the first agarose layer and allowed to solidify under a clean cover glass. After removing the cover glass 100 ml of 0.5% low melting point agarose was added and allowed to solidify in a chilled condition. After the cover glass was removed, the slides were gently immersed in a freshly prepared cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO and 1% Triton X-100, pH adjusted to 10 with NaOH) and kept at 4 1C in the dark for 1 h. The slides were placed on the horizontal gel electrophoresis unit filled with fresh, cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13.5) for 20 min. Electrophoresis was conducted for the next 20 min at 18 V (1.0 V/cm, 250 mA). The slides were then drained, placed on a tray and flooded slowly with three changes of neutralization buffer (0.4 M Tris–HCl, pH 7.5), each for 5 min. The slides were stained with ethidium bromide (10 mg/ml), covered with a cover glass, and analyzed within 1 h
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at 100 magnification using a fluorescent microscope (Motic BA400, Germany) with green filter. The photograph was taken through the attached digital camera. The comet length–width ratio (L:W), tailed cell % (TC%) was recorded by randomly counting about 100 cells per slide, considering no overlap of counting, using Motic Images Plus 2.0 software. 2.14. Statistics Values were shown as mean7SE. Statistical analysis was done by Student’s t-test and one way Anova, Po0.05 was considered significant.
4.5
Control Ara-C TSE
4
3.89
3.5 Cell count x 105
392
3 2.34
2.5 2
1.82
1.5 0.67 0.59
1
3. Results
0.6 0.52
0.61 0.54
0.5
3.1. IC50 value of TSE
0 24 h
(A)
At 95% of fiducial probabilities the confidence intervals of IC50 value of TSE in U937 cells was 55.0–66.2 mg/ml. In K562 cells at 95% of fiducial probabilities the confidence intervals of IC50 value of TSE was 81.1–92.2 mg/ml.
2.5
48 h Control IM TSE
72 h 2.33
2
3.2. U937 and K562 cell growth inhibition TSE (100 mg/ml) produced time-dependent inhibition of U937 cell growth as compared to control untreated cells. At 24, 48 and 72 h standard Ara-C (100 mg/ml) showed 63.2%, 73.9% and 84.6% inhibition of U937 cell growth. Whereas TSE (100 mg/ml) at 24, 48 and 72 h produced 67.6%, 76.9% and 86.6% inhibition of U937 cell growth, respectively. In K562 cells at 24, 48 and 72 h of treatment standard imatinib mesylate (100 mg/ml) showed 61.1%, 73.3% and 82.0% inhibition of cell growth. However, TSE produced 58.8%, 71.0% and 79.0% inhibition of K562 cell growth at 24, 48, 72 h, respectively (Fig. 1, Table 1). 3.3. MTT assay It was observed at 24 h that, standard Ara-C (100 mg/ml) and TSE (100 mg/ml) showed 35.3% and 69.3% reduction in OD value, respectively, as compared with untreated control U937 cells. The OD of control, Ara-C treated and TSE treated U937 cells were 2.6570.13, 1.7270.10 and 0.8270.03, respectively (Fig. 2). In K562 cell line at 24 h of observation, standard Imatinib mesylate (100 mg/ml) and TSE (100 mg/ml) treated groups showed 33.6% and 44.1% reduction in OD value as compared with untreated
Cell count x 105
1.76
1.5
1.31
1 0.54 0.51
0.51 0.47
0.49 0.42
0.5
0 (B)
24 h
48 h
72 h
Fig. 1. TSE-induced inhibition of U937 and K562 cell growth. Panel-A: U937 cells and Panel-B: K562 cells. Bar diagram represents the cell count ( 105) at 24, 48 and 72 h of treatment in respective control, standard Ara-C/IM and TSE-treated groups. Error bars represent SEM and the data shown above the bar represent mean data. TSE showed significant reduction in cell growth (Po0.001) in all the cell groups at 24, 48 and 72 h.
control group. The OD of control, imatinib mesylateand TSE-treated U937 cells were 2.2170.08, 1.4770.04 and 1.2470.02, respectively (Fig. 2). 3.4. PCNA expression Percentage positivity of PCNA expression in U937 cells in control, standard (Ara-C, 100 mg/ml)
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Table 1 TSE-induced U937 and K562 cell growth inhibition Type of cells
Time (h)
Control (cell count 105)
Ara-C/IM (100 mg/ml) (cell count 105)
TSE (100 mg/ml) (cell count 105)
U937 cells
24 48 72
1.8270.026 2.3470.066 3.8970.085
0.6770.014 0.6170.013 0.6070.011
0.5970.009 0.5470.010 0.5270.014
K562 cells
24 48 72
1.3170.025 1.7670.027 2.3370.026
0.5170.011 0.4770.007 0.4270.012
0.5470.015 0.5170.010 0.4970.014
Values as Mean7SEM. po0.001, significant.
80
3 2.65
Control Standard TSE
2.5
70
TSE
60
1.72 1.47
1.5
1.24
% Positivity
OD (570 nm)
Standard
59
2.11 2
Control
68
50
57 47
43 38
40 30
0.82
1
20 10
0.5
0 U937
0 U937
K562
Fig. 2. Effect of TSE on the MTT assay of U937 and K562 cells. Reduction in MTT values are significantly different (Po0.001) in TSE-treated groups of U937 and K562 cells from the respective controls. Error bars represent SEM. Data shown above the bars are mean OD of each group.
and TSE (IC50 dose) treated groups were found to be 6871.6%, 4371.8% and 5971.3%, respectively, at 24 h of treatment. In K562 cell line at the same treatment period, the untreated control, standard (imatinib mesylate, 100 mg/ml) and TSE (IC50 dose) treated groups showed percentage positivity of PCNA expression as 5772.1%, 3872.0% and 4771.6%, respectively. The significant reduction in PCNA expression in U937 and K562 cells provide clue for the involvement of TSE in the cell proliferation control machinery which may be decelerated due to treatment (Fig 3).
K562
Fig. 3. Effect of TSE on the PCNA expression of U937 and K562 cells. The data shown above the bars represent the mean of percentage positivity of PCNA expression. Error bars represent SEM. PCNA of TSE-treated cell groups are significantly different (Po0.01) from respective control groups.
3.5. Cell cycle analysis Flow cytometric data on cell cycle phase distribution at 24 h of culture, with TSE (IC50 dose) treated U937 cells showed 66%, 23% and 11% of G1, S and G2 phase cells in the cell cycle. Whereas control U937 cells showed 57%, 29% and 16% of G1, S and G2 phase cells (Fig 4). In K562 cells, TSE (IC50 dose) showed 71%, 22% and 7% cells at G1, S and G2 phase cells, where as control K562 cells showed 61%, 23% and 16% of G1, S and G2 phase cells (Fig 4). The standard Ara-C in U937 cell and
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394
66
70
Control Ara-C
62 60
57
TSE
K562 cells (Fig. 5). The standards were taken in this experiment as positive control. 3.7. Confocal microscopy
Cells (%)
50
U937 and K562 cells treated with TSE (IC50 dose) showed nuclear fragmentation and marginalization of fragmented nuclei towards the membrane (Fig 6).
40 29 29 30
23 16
20
3.8. Scanning electron microscopy 9
11
The morphological changes including membrane blebbing, membrane perforation and cell surface shrinkage were observed in U937 and K562 cells of TSE treated group (Fig 7).
10 0 G1
S
G2
Cell cycle phase
(A) 80
74
3.9. Alkaline comet assay 71
Control IM TSE
70 61 60
Cells (%)
50 40 30
23
20 22 16
20
6
10
7
0 G1 (B)
S Cell cycle phase
G2
Fig. 4. Effect of TSE on the cell cycle phase distribution of U937 and K562 cells. Panel-A: U937 cells and Panel-B: K562 cells. The mean percentage cells in G1, S and G2 phases of cell cycle were represented in both Panel A and B.
imatinib mesylate in K562 cells also showed G1 phase arrest in the cell cycle. 3.6. Annexin-V binding study The U937 control cell showed 4.14% binding, whereas TSE (IC50 dose) treated cells bound about 14.44%. On the other hand, K562 control cell showed 3.33% binding and TSE (IC50)-treated cells showed 21.51% annexin binding. Standard Ara-C and imatinib mesylate-induced Annexin-V bindings were 19.43% and 21.86%, respectively, in U937 and
The mean length-to-width ratio (L:W) of the DNA mass observed in TSE-treated U937 was significantly greater (Po0.001) compared with the normal control cells. Similarly, the mean frequency of TC% was (84.070.17) in TSE-treated U937 cells, which was significantly different (Po0.001) from the normal control (11.270.06). The mean L:W of the DNA mass in TSE (IC50 dose) treated K562 was significantly greater (Po0.001) as compared with the normal control cells. Similarly, the mean frequency of TC% was (89.270.34) in TSE-treated K562 cells was significantly different (Po0.001) from the normal control (9.370.11) (Fig 8, Table 2). 3.10. Viability of normal human peripheral blood mononuclear cells At 24 h of PBMNCs culture the viable cell count in control, imatinib mesylate (100 mg/ml), Ara-C (100 mg/ml) and TSE (100 mg/ml)-treated cells were (1.5870.06) 105, (1.1970.04) 105, (1.2870.04) 105 and (1.2670.04) 105, respectively. The viable cell count in Imatinib mesylate, Ara-C and TSE-treated cells decreased significantly (Po0.001) by 24.7%, 19.0% and 20.3%, respectively, as compared with control. 3.11. Cytotoxicity study of normal human peripheral blood mononuclear cells MTT assay of PBMNCs after 24 h of culture in presence and absence of Imatinib mesylate (100 mg/ml), Ara-C (100 mg/ml) and TSE (100 mg/ml)
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Fig. 5. Effect of TSE on the Annexin-V binding of U937 and K562 cells. Left panel: U937 cells (A: control, B: Ara-C standard, C: TSE-treated). Right panel: K562 cells (D: control, E: imatinib mesylate standard, F: TSE-treated). The flow cytometric analysis of percentage of Annexin-V bindings in each group are shown in the figure of respective cell groups.
revealed that the optical density of color formazan were significantly (Po0.001) reduced by 22.0%, 16.7% and 19.6%, respectively, as compared with control cells. The optical densities of color formazan in MTT assay in control, imatinib mesylate, Ara-C and TSE-treated PBMNCs were found to be 0.9970.01, 0.7770.01, 0.8270.02 and 0.7970.01, respectively. 4. Discussion It is now well established that the reduced capacity of tumor cells undergoing cell death
through apoptosis, plays a key role both in the pathogenesis and therapeutic failure of cancer. The fate of cancer cells primarily depends on the proliferation control i.e., DNA replication and apoptosis induction through the coordinated cell cycle regulation (Lam et al., 2004). In the present study, TSE showed time-dependent inhibition of U937 and K562 cell growth and proliferation. TSEinduced U937 and K562 cell growth inhibition and reduction in MTT values were correlated with the reduction in cell proliferation and cytotoxic effect of TSE. The reduction OD value has a direct
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Fig. 6. Confocal laser scanning photomicrograph of U937 and K562 cells. Upper panel: U937 (A, Control and B, TSE-treated) cells. Lower panel: K562 (C, Control and D TSE-treated) cells. * a, Marginalization of fragmented nuclei, and * b, Fragmented nuclei Magnification (1000 ).
correlation with the growth inhibitory rate and inverse relation with proliferative rate (Andrew et al., 2002). MTT is a water-soluble tetrazolium salt, which is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by mitochondrial succinate dehydrogenase. The disruption of mitochondrial dehydrogenase system due to the mitochondrial dysfunction or apoptosis, may reduce the color formazan production in the MTT assay. Cytotoxic and antiproliferative drugs may disrupt this system, which was reflected in the MTT assay. TSE suppressed the PCNA expression in U937 and K562 cells. PCNA is centrally involved in the regulation and coordination of cell cycle progression and DNA replication. It stimulates the activity of DNA polymerase d and increases its processivity by acting as a clamp platform that slides along the DNA template (Kontopidis et al., 2005). The suppression of PCNA expression as well as the cell cycle arrest primarily at G1 phase in U937
and K562 cells were correlated with the cell growth inhibition and apoptosis induction by the TSE. The antiproliferative effect of TSE and the cell cycle arrest with the simultaneous suppression of PCNA expression may explain the fact that TSE halts the cell cycle for the prevention of further cell proliferation and execution of apoptotic signaling. At 24 h, TSE-induced apoptosis in U937 and K562 cells were detected using Annexin-V-FITC binding by flow cytometry. Annexin-V binding is based on the transposition of phosphatidyl serine from the inner to the outer face of cell membrane during the early stages of apoptosis (Vermes et al., 1995). The nuclear fragmentation and formation of apoptotic bodies in U937 and K562 cells indicated the apoptogenic nature of TSE. TSE deformed the cancer cell membrane architecture and produced membrane blebbing and perforation, which is the morphological marker for apoptosis. The blebbing and pore formation may be due to the disruption of
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Fig. 7. Scanning electron photomicrograph of U937 and K562 cells. Left panel: Control (A, U937 and C, K562 ). Right panel: TSE-treated (B, U937 and D, K562) groups. * a, Membrane blebbing, * b, Membrane perforation. Magnification (3000 ).
cellular cytoskeleton element due to TSE. TSE induced DNA fragmentation was also reflected in single-cell gel electrophoresis. TSE produced significant increase in L:W in U937 and K562 cells. This is due to the movement of fragmented DNA mass of the apoptotic cells towards the anode during electrophoresis in alkaline condition. There is a direct relation of increase in L:W of DNA mass with the extent of DNA fragmentation (Singh et al., 1988). This assay is able to detect very short-lived primary DNA lesions such as single-strand break as well as double-strand breaks in the DNA of individual cells (Hartmann et al., 2004). Apoptosis leads to cytoskeletal disruption, cell shrinkage, membrane blebbing, nuclear fragmentation and disruption of DNA into fragments (Fairbairn et al., 1996). Recent observation on the studies of Chinese toad (B. b. gargarizans) skin preparation (Chan Sue) with flow cytometry, agarose gel electrophoresis of
DNA and fluorescent staining in human bladder carcinoma cell line, indicated its apoptogenic nature (Ko et al., 2005). Chan Sue induced apoptosis in human leukemic cells (U937) was also reported (Watabe et al., 1997). Genomic instability due to DNA or nuclear fragmentation usually induces the activation of p53, resulting in growth arrest or apoptosis (Dasika et al., 1999). Ablation of PCNA expression or function in cells under proliferative stimuli, appears to constitute an apoptotic trigger. The tumor-suppressor protein p21 modulates cell cycle progression by direct binding to PCNA and also by inhibiting cyclin-dependent kinases (CDKs). Arresting cells in G1, p21 can inhibit Cdc2–cyclin B complexes and proliferating cell nuclear antigen preventing interaction of the latter with other components of the DNA polymerase complex (Vidal and Koff, 2000; Waga et al., 1994). At the cellular level the competition between p21 and DNA replication factors for binding to PCNA is believed
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Fig. 8. Single cell gel electrophoresis (comet assay) of U937 and K562 cells. Left panel: control (A, U937 and C, K562). Right panel: TSE-treated (B, U937 and D, K562). * Tailed cells, Magnification (100 ).
Table 2 TSE-induced DNA fragmentation in U937 and K562 cells in comet assay Experimental group
Tailed cells (%)
Length–width ratio of DNA mass
Control U937 cells TSE treated (IC50 Dose) U937 cells Control K562 cells TSE treated (IC50 Dose) K562 cells
11.270.06 84.070.17 9.370.11 89.270.34
1.1270.048 2.2170.106 1.0770.078 2.1670.110
Values as mean7SEM. po0.001, significant.
to be the mechanism through which DNA synthesis is inhibited (Kontopidis et al., 2005). In the present study, the Annexin-V binding, DNA and nuclear fragmentation, membrane blebbing and the G1 arrest, all support the apoptogenic nature of TSE, whereas cell growth inhibition, suppression of PCNA expression and decreased absorbance of TSE-treated U937 and K562 cells in MTT assay indicated the antiproliferative and cytotoxic activity of TSE. However, TSE-induced growth inhibition and cytotoxicity in U937 and K562 cancer cells were found to be higher than that of the normal PBMNCs.
It may be concluded that, Indian TSE possesses potent antineoplastic agent, which is antiproliferative, cytotoxic and apoptogenic against human myeloid leukemic cells (U937 and K562). Isolation/purification of the active anticancer molecule from TSE is in progress. Acknowledgments The present study was partially financed by the University Grants Commission, New Delhi Government of India [Ref.: UGC/417/Major Research(Sc.)03; dt. 31.07.2003)]. Imatinib mesylate
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was a gift from Natco Pharma Ltd., Hyderabad, India.
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Toxicon 48 (2006) 388–400 www.elsevier.com/locate/toxicon
Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract on U937 and K562 cells B. Giria, A. Gomesb, A. Debnathb, A. Sahaa, A.K. Biswasa, S.C. Dasguptaa, A. Gomesa, a
Laboratory of Toxinology and Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata 700 009, India b New Drug Development Division, Indian Institute of Chemical Biology, 4, Raja Subodh Mallik Road, Kolkata 700 032, India Received 20 February 2006; received in revised form 20 June 2006; accepted 22 June 2006 Available online 29 June 2006
Abstract The antiproliferative, cytotoxic and apoptogenic activities of Bufo melanostictus (Indian common toad) skin extract (TSE) on U937 and K562 leukemic cell line has been investigated. TSE significantly (Po0.001) reduced the time-dependent cell proliferation and decreased MTT values in U937 and K562 cells. TSE (IC50 doses) suppressed the proliferating cell nuclear antigen expression in both the cells. It was demonstrated that, TSE (IC50 doses) primarily arrested the U937 and K562 cells at G1 phase of the cell cycle. Confocal microscopy showed the altered fragmented nuclei and apoptotic bodies formation in TSE (IC50 doses) treated U937 and K562 cells. Membrane blebbing, cell surface shrinkage and perforation were observed through scanning electron microscope. TSE-induced DNA fragmentation in U937 and K562 cells was reflected in single-cell gel electrophoresis. TSE significantly (Po0.001) increase the length–width ratio of DNA mass as compared to control in comet assay. The flow cytometric analysis of annexin-V binding to the cancer cells further supported the apoptotogenic activity of TSE. The effect of TSE on normal human peripheral blood mononuclear cells viability and cytotoxicity was studied in culture and found to be less cytotoxic than on the U937 and K562 cells. The findings from the present study suggested that TSE might possess potent antineoplastic agent having antiproliferative, cytotoxic and apoptogenic activity against U937 and K562 myeloid leukemic cells. r 2006 Elsevier Ltd. All rights reserved. Keywords: Toad skin; Skin extract; Anticancer activity; Cytotoxic agent; U937 cell; K562 cell
1. Introduction Cancer is a complex multifactorial disease of the cell. Cancer development and progression are Corresponding author. Tel.: +91 33 2244 4755; fax: +91 33 2351 9755. E-mail address: [email protected] (A. Gomes).
0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.06.011
dependent on cellular accumulation of various genetic and epigenetic events (Blagosklonny, 2005). Radiotherapy, chemotherapy and surgical measure are the key tools for cancer treatment. In the hematopoetic malignancies, chemotherapeutic approaches are widely applied in practice. Nowadays, several therapeutic approaches have been taken to overcome the complexities of different
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cancers. Drug discovery against cancer is ventured throughout the world especially from the natural products. It is evident to note that amphibians like frog and toad possess different bioactive substances in their skin (Duellman and Trueb, 1986). Amphibian skin is a morphologically, biochemically and physiologically complex organ, which fulfils a wide range of functions necessary for the amphibian survival, including respiration, water regulation, antipredator, antimicrobial defense, excretion, temperature control, etc., (Clarke, 1997). Toads, particularly members of the genus Bufo, are identified as a particularly convenient and useful source of granular gland secretions, which commonly contain biogenic amines, bufodienolides, alkaloids and steroids, peptides and proteins (Cei et al., 1967; Clarke, 1997; Daly et al., 2004; Maciel et al., 2003; Steyn and Van Heerden, 1998). In the Chinese traditional medicine, amphibian’s skin extract had been used for the alleviation of human sufferings (Clarke, 1997; Ko et al., 2005). Chan Sue, the Chinese toad (Bufo bufo gargarizans) skin extract preparation was used in the treatment of various diseases, like cancer, arrhythmia and other heart diseases. (Datta and Dasgupta, 2002; Bhuiyan et al., 2003; Nogawa et al., 2001). A number of research is going on in other parts of the world regarding the anticancer activities of different toad and frog skin components; but few scientific information are available on the Indian toad and frog skin bioactive compound. Earlier investigation from this laboratory revealed that Indian toad (Bufo melanostictus) skin extract is pharmacologically potent having immunomodulatory and antineoplastic activity on Ehrlich ascites carcinoma bearing mice (Das et al., 1998a; Das et al., 1998b). The present communication is an approach to study the antiproliferative, cytotoxic and apoptogenic activities of Indian toad (B. melanostictus) skin extract on U937 and K562 cells. 2. Materials and methods
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mesylate (Natco, India), low melting point agarose (Promega, USA), methanol (Spectrochem, India), MTT (Sigma, USA), normal melting point agarose (Promega, USA), osmium tetraoxide (Sigma, USA) Penicillin–streptomycin (Biowest, Germany) proliferating cell nuclear antigen (PCNA) antibody (Santa Cruz Biotechnology, Inc., USA) phytohemagglutinin (Sigma-Aldrich, USA), polylysine (Sigma, USA), propidium iodide (Sigma, USA), RPMI (GIBCO BRL, USA), sodium cacodylate (SRL, India), Triton X-100 (SRL, India), trypan blue (SRL, India). 2.2. Collection and preparation of toad skin extract (TSE) Adult toads (B. melanostictus, Schneider) of both sexes (80710 g) were collected commercially from Ms. Reeta Ghosh & Company, Calcutta, India. Skin was collected from freshly pithed toad, washed in distilled water, weighed and immersed in 99.5% methanol at room temperature for 30–40 days. The supernatant was collected and filtered. It was evaporated to dryness by rotary evaporator and the extract was kept at room temperature in a desiccator. The yield of the extract was 1.370.46% of the skin and expressed in terms of dry weight. The extract (TSE) was suspended in RPMI medium for the experiments. 2.3. Human myeloid leukemic cells and culture U937 and K562 cell lines were procured from the National Facility for Animal Tissue and Cell culture, Pune, India. Cell line was used after 17 passages. U937 and K562 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (heat inactivated), penicillin (100 units/ml), streptomycin (100 mg/ml) and gentamicin (100 mg/ml). Cell lines were maintained in a humidified incubator with 5% CO2 and 95% air at 37 1C. It was subcultured weekly at an initial density of 1 106 cells/ ml and maintained.
2.1. Chemicals used Annexin-V antibody (Sigma, USA), Ara-C (Sigma, USA), Dimethyl sulfoxide (DMSO) (SRL, India), EDTA (Sigma, USA), ethidium bromide (Sigma, USA), fetal bovine serum (GIBCO, BRL, USA), gentamicin (Nicholas, India), glutaraldehyde (Merck, India), HEPES (Sigma, USA), heparin (Sigma, USA), histopaque (Sigma, USA), imatinib
2.4. Normal human peripheral blood mononuclear cell culture and its viability and cytotoxicity studies After informed consent, 5 ml of blood was drawn (venipuncture) aseptically from healthy human volunteers (25–30 years of age) and transferred into sterile tubes containing 10 U/ml heparin inside a laminar flow hood. Heparinized blood was diluted
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with equal volume of normal saline. About 10 ml of diluted blood was carefully layered on 3 ml of histopaque. It was then centrifuged at 1500 rpm for 20 min at room temperature. Peripheral blood mononuclear cells (PBMNC) were collected from histopaque–plasma interface and washed twice with normal saline. The cell pellet was resuspended in culture medium (as mentioned in Section 2.3.) with density of 1 106 cells/ml and cultured in a CO2 incubator with 95% O2 and 5% CO2 at 37 1C for 24 h in presence and absence of TSE (100 mg/ml) and standard anticancer drugs imatinib mesylate (100 mg/ml) and Ara-C (100 mg/ml). PBMNCs were stimulated with 2.5 mg/ml of phytohemoagglutinin (Kondala et al., 2005). Cell viability was determined using Trypan blue (0.4%) dye exclusion procedure and viable cells were counted in a phase contrast microscope (Olympus CK40) and percentage (%) inhibition of viable cells was calculated as compared with control. The extent of cytotoxicity was analyzed and expressed as % inhibition of OD values in MTT assay (as mentioned in Section 2.7.) (Kawada et al., 2002). 2.5. Determination of IC50 The 50% of inhibition concentration (IC50) values of TSE in U937 and K562 cells were determined using different concentrations (25, 50, 75, 100, 125, 150 mg/ml) of the extract at 24 h. The respective viable cells were counted in the phase contrast microscope using Trypan blue (Agrawal et al., 1989). The respective concentration of extract was converted to their respective log dose. The percentage of cell death was converted to Probit value in respective dose concerned. The graph was plotted with the different log dose of the extract against the respective Probit value using Origin 6.0 Professional software. The IC50 value was noted. The above mentioned procedure were repeated four times and the confidence intervals of IC50 values were calculated.
was incubated in a humidified 5% CO2 incubator for 24, 48 and 72 h at 37 1C. The number of viable cells was determined by the Trypan blue dye exclusion test and the percentage inhibition of cell growth was calculated as compared with control (Banks-Schlegel and Quintero, 1986; Agrawal et al., 1989). 2.7. MTT assay MTT assay was done according to Kawada et al., (2002). One hundred microliters of myeloid leukemic cells (U937 and K562) suspension in RPMI 1640 and supplemented with 10% foetal calf serum containing 1 105 cells were added to each well of a 96-well microtiter plate separately. Cancer cells were cultured in presence and absence of TSE (100 mg/ml). Plates were incubated at 37 1C in a humidified 5% CO2 incubator for 24 h. Twenty microliters of MTT (5 mg/ml) was then added in each well and allowed to incubate at 37 1C in 5% CO2 incubator for 3 h. 100 ml DMSO was added to each well to dissolve the formazan crystal formed. The OD was recorded at 570 nm with microplate reader (Merck-MIOS Mini, Model No. 309). Growth inhibitory rate (%) was expressed as ¼ [1(OD of treated/OD of control)] 100. 2.8. Proliferating cell nuclear antigen (PCNA) expression After 24 h of TSE treatment (IC50 doses), U937 and K562 cells were mechanically and enzymatically dissociated to obtain bare nuclei, treated with RNAase, then stained with propidium iodide. The nuclei suspensions were acquired and analyzed on TM Becton Dickinson (BD LSR three laser Flow Cytometer, using 488/UV/HeNe 488 nm Argon ion/ 325 nm HeCd/633 nm laser) and internal signals from the blue photomultiplier transducer. A total events of 10,000 cells were collected for each sample. PCNA expression was done using antihuman rabbit PCNA antibody and expressed as percentage positivity (Giordano et al., 1991).
2.6. Cell growth inhibition study 2.9. Cell cycle analysis To observe cell growth inhibitory effects, 100 ml of myeloid leukemic cell (U937 and K562) suspension containing 1 106 cells/ml in culture medium in each well was added in 96-well microtitre plate separately. The TSE was dissolved in sterile RPMI medium and cancer cells were cultured in presence and absence of TSE (100 mg/ml). The culture plate
For the analysis of cell cycle phase distribution of DNA, respective control, standard and TSE-treated cancer cells (1 106 each) were washed with PBS after 24 h of TSE treatment (IC50 doses) and fixed in 70% ethanol and stained with propidium iodide (100 mg/ml in 0.1% sodium citrate containing 0.1%
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Triton X-100) after RNAase (10 mg/ml) treatment (Lim et al., 1999). The cell cycle distribution was determined by flow cytometry (Becton Dickinson, TM BD LSR three-laser Flow Cytometer) using CellQuest software. Fluorescence detector equipped with 488 nm Argon laser light source and 623 nm band pass filter. Total of 10,000 cells were acquired for analysis. 2.10. Annexin-V binding study U937 and K562 cells (1 106) were cultured in presence and absence of TSE (IC50 doses) for 24 h. The cells were then washed with phosphate buffer saline and centrifuged at 1500 rpm at 4 1C. The cell pellet was washed twice with 1X Annexin–Hepes buffer and resuspended in 100 ml of this buffer. Annexin-V antibody (final concentration of 0.2 mg/ml) was added to this cell suspension and incubated for 30 min in ice in the dark room. Then it was washed again with 1X Annexin–Hepes buffer and fixed with 1% paraformaldehyde at 4 1C. The cells were analyzed within 3–4 h by flow cytometry (Becton Dickinson, FACS Calibur) with Cell Quest software. Flow cytometer was set for collecting data of 10,000 cells in each group (Vermes et al., 1995). 2.11. Confocal microscopy Cancer cells (1 105) were harvested and centrifuged at 4000 rpm for 4 min and washed with PBS after 24 h of TSE (IC50 doses) treatment. The pellet was then suspended in 100 ml of PBS. Ten microliters of cell suspension was spotted on polylysinecoated coverslip and incubated at 4 1C for 30 min. It was then washed thrice with PBS. Methanol/ acetone (1:1) mixture was added on it and kept for 5 min. It was again washed with PBS. Ten microliters of ethidium bromide (100 mg/ml) containing 10% glycerol was taken on the fixed cells smear of the coverslip. It was then covered with a prepolylysine-coated coverslip. The prepared cell sandwich was kept in a moist petri dish overnight before analysis. It was then analyzed in a laser scanning confocal microsope (Leica Model SP2) (Guan et al., 2004). 2.12. Scanning electron microscopy U937 and K562 cells (1 105) were cultured in presence and absence of TSE (IC50 doses) for 48 h. The cells were then washed twice with phosphate
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buffer saline (pH 7.2) and centrifuged at 1500 rpm at 4 1C. It was fixed with 2.5% glutaraldehyde at 4 1C for 3 h in dark. The samples were again washed with PBS and post-fixed in 1% OsO4 in a cacodylate buffer for 1 h. Finally, cells were dehydrated in ascending grade of ethanol and embedded in polylysine-coated thick cover glass. The prepared cells on the cover glass were then gold coated and analyzed at 15 KV accelerating voltage by Leica (Model S440) scanning electron microscope (Nottola et al., 2005). 2.13. Single-cell gel electrophoresis (alkaline comet assay) Comet assay of U937 and K562 cells was performed under alkaline condition following method of Singh et al., (1988) with minor modifications. U937 and K562 cells (1 105) were cultured in presence and absence of TSE (IC50 doses) for 48 h. The cells were then washed twice with cold PBS by centrifuging at 1500 rpm for 5 min in cold centrifuge. Concentration (1–2 104/10 ml) of different cell groups was standardized by changing the dilution of cell suspension. Microscope slides were covered with 400 ml of 0.75% normal melting point agarose in PBS pre-warmed to 50 1C. A cover glass was placed over the agarose solution, and the agarose allowed to solidify. The coverglass was then removed, and 85 ml of cell–agarose suspension (10 ml of cell suspension containing about 104 cells was mixed with 75 ml of 0.5% low melting point agarose, in PBS) was placed over the first agarose layer and allowed to solidify under a clean cover glass. After removing the cover glass 100 ml of 0.5% low melting point agarose was added and allowed to solidify in a chilled condition. After the cover glass was removed, the slides were gently immersed in a freshly prepared cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO and 1% Triton X-100, pH adjusted to 10 with NaOH) and kept at 4 1C in the dark for 1 h. The slides were placed on the horizontal gel electrophoresis unit filled with fresh, cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13.5) for 20 min. Electrophoresis was conducted for the next 20 min at 18 V (1.0 V/cm, 250 mA). The slides were then drained, placed on a tray and flooded slowly with three changes of neutralization buffer (0.4 M Tris–HCl, pH 7.5), each for 5 min. The slides were stained with ethidium bromide (10 mg/ml), covered with a cover glass, and analyzed within 1 h
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at 100 magnification using a fluorescent microscope (Motic BA400, Germany) with green filter. The photograph was taken through the attached digital camera. The comet length–width ratio (L:W), tailed cell % (TC%) was recorded by randomly counting about 100 cells per slide, considering no overlap of counting, using Motic Images Plus 2.0 software. 2.14. Statistics Values were shown as mean7SE. Statistical analysis was done by Student’s t-test and one way Anova, Po0.05 was considered significant.
4.5
Control Ara-C TSE
4
3.89
3.5 Cell count x 105
392
3 2.34
2.5 2
1.82
1.5 0.67 0.59
1
3. Results
0.6 0.52
0.61 0.54
0.5
3.1. IC50 value of TSE
0 24 h
(A)
At 95% of fiducial probabilities the confidence intervals of IC50 value of TSE in U937 cells was 55.0–66.2 mg/ml. In K562 cells at 95% of fiducial probabilities the confidence intervals of IC50 value of TSE was 81.1–92.2 mg/ml.
2.5
48 h Control IM TSE
72 h 2.33
2
3.2. U937 and K562 cell growth inhibition TSE (100 mg/ml) produced time-dependent inhibition of U937 cell growth as compared to control untreated cells. At 24, 48 and 72 h standard Ara-C (100 mg/ml) showed 63.2%, 73.9% and 84.6% inhibition of U937 cell growth. Whereas TSE (100 mg/ml) at 24, 48 and 72 h produced 67.6%, 76.9% and 86.6% inhibition of U937 cell growth, respectively. In K562 cells at 24, 48 and 72 h of treatment standard imatinib mesylate (100 mg/ml) showed 61.1%, 73.3% and 82.0% inhibition of cell growth. However, TSE produced 58.8%, 71.0% and 79.0% inhibition of K562 cell growth at 24, 48, 72 h, respectively (Fig. 1, Table 1). 3.3. MTT assay It was observed at 24 h that, standard Ara-C (100 mg/ml) and TSE (100 mg/ml) showed 35.3% and 69.3% reduction in OD value, respectively, as compared with untreated control U937 cells. The OD of control, Ara-C treated and TSE treated U937 cells were 2.6570.13, 1.7270.10 and 0.8270.03, respectively (Fig. 2). In K562 cell line at 24 h of observation, standard Imatinib mesylate (100 mg/ml) and TSE (100 mg/ml) treated groups showed 33.6% and 44.1% reduction in OD value as compared with untreated
Cell count x 105
1.76
1.5
1.31
1 0.54 0.51
0.51 0.47
0.49 0.42
0.5
0 (B)
24 h
48 h
72 h
Fig. 1. TSE-induced inhibition of U937 and K562 cell growth. Panel-A: U937 cells and Panel-B: K562 cells. Bar diagram represents the cell count ( 105) at 24, 48 and 72 h of treatment in respective control, standard Ara-C/IM and TSE-treated groups. Error bars represent SEM and the data shown above the bar represent mean data. TSE showed significant reduction in cell growth (Po0.001) in all the cell groups at 24, 48 and 72 h.
control group. The OD of control, imatinib mesylateand TSE-treated U937 cells were 2.2170.08, 1.4770.04 and 1.2470.02, respectively (Fig. 2). 3.4. PCNA expression Percentage positivity of PCNA expression in U937 cells in control, standard (Ara-C, 100 mg/ml)
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Table 1 TSE-induced U937 and K562 cell growth inhibition Type of cells
Time (h)
Control (cell count 105)
Ara-C/IM (100 mg/ml) (cell count 105)
TSE (100 mg/ml) (cell count 105)
U937 cells
24 48 72
1.8270.026 2.3470.066 3.8970.085
0.6770.014 0.6170.013 0.6070.011
0.5970.009 0.5470.010 0.5270.014
K562 cells
24 48 72
1.3170.025 1.7670.027 2.3370.026
0.5170.011 0.4770.007 0.4270.012
0.5470.015 0.5170.010 0.4970.014
Values as Mean7SEM. po0.001, significant.
80
3 2.65
Control Standard TSE
2.5
70
TSE
60
1.72 1.47
1.5
1.24
% Positivity
OD (570 nm)
Standard
59
2.11 2
Control
68
50
57 47
43 38
40 30
0.82
1
20 10
0.5
0 U937
0 U937
K562
Fig. 2. Effect of TSE on the MTT assay of U937 and K562 cells. Reduction in MTT values are significantly different (Po0.001) in TSE-treated groups of U937 and K562 cells from the respective controls. Error bars represent SEM. Data shown above the bars are mean OD of each group.
and TSE (IC50 dose) treated groups were found to be 6871.6%, 4371.8% and 5971.3%, respectively, at 24 h of treatment. In K562 cell line at the same treatment period, the untreated control, standard (imatinib mesylate, 100 mg/ml) and TSE (IC50 dose) treated groups showed percentage positivity of PCNA expression as 5772.1%, 3872.0% and 4771.6%, respectively. The significant reduction in PCNA expression in U937 and K562 cells provide clue for the involvement of TSE in the cell proliferation control machinery which may be decelerated due to treatment (Fig 3).
K562
Fig. 3. Effect of TSE on the PCNA expression of U937 and K562 cells. The data shown above the bars represent the mean of percentage positivity of PCNA expression. Error bars represent SEM. PCNA of TSE-treated cell groups are significantly different (Po0.01) from respective control groups.
3.5. Cell cycle analysis Flow cytometric data on cell cycle phase distribution at 24 h of culture, with TSE (IC50 dose) treated U937 cells showed 66%, 23% and 11% of G1, S and G2 phase cells in the cell cycle. Whereas control U937 cells showed 57%, 29% and 16% of G1, S and G2 phase cells (Fig 4). In K562 cells, TSE (IC50 dose) showed 71%, 22% and 7% cells at G1, S and G2 phase cells, where as control K562 cells showed 61%, 23% and 16% of G1, S and G2 phase cells (Fig 4). The standard Ara-C in U937 cell and
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394
66
70
Control Ara-C
62 60
57
TSE
K562 cells (Fig. 5). The standards were taken in this experiment as positive control. 3.7. Confocal microscopy
Cells (%)
50
U937 and K562 cells treated with TSE (IC50 dose) showed nuclear fragmentation and marginalization of fragmented nuclei towards the membrane (Fig 6).
40 29 29 30
23 16
20
3.8. Scanning electron microscopy 9
11
The morphological changes including membrane blebbing, membrane perforation and cell surface shrinkage were observed in U937 and K562 cells of TSE treated group (Fig 7).
10 0 G1
S
G2
Cell cycle phase
(A) 80
74
3.9. Alkaline comet assay 71
Control IM TSE
70 61 60
Cells (%)
50 40 30
23
20 22 16
20
6
10
7
0 G1 (B)
S Cell cycle phase
G2
Fig. 4. Effect of TSE on the cell cycle phase distribution of U937 and K562 cells. Panel-A: U937 cells and Panel-B: K562 cells. The mean percentage cells in G1, S and G2 phases of cell cycle were represented in both Panel A and B.
imatinib mesylate in K562 cells also showed G1 phase arrest in the cell cycle. 3.6. Annexin-V binding study The U937 control cell showed 4.14% binding, whereas TSE (IC50 dose) treated cells bound about 14.44%. On the other hand, K562 control cell showed 3.33% binding and TSE (IC50)-treated cells showed 21.51% annexin binding. Standard Ara-C and imatinib mesylate-induced Annexin-V bindings were 19.43% and 21.86%, respectively, in U937 and
The mean length-to-width ratio (L:W) of the DNA mass observed in TSE-treated U937 was significantly greater (Po0.001) compared with the normal control cells. Similarly, the mean frequency of TC% was (84.070.17) in TSE-treated U937 cells, which was significantly different (Po0.001) from the normal control (11.270.06). The mean L:W of the DNA mass in TSE (IC50 dose) treated K562 was significantly greater (Po0.001) as compared with the normal control cells. Similarly, the mean frequency of TC% was (89.270.34) in TSE-treated K562 cells was significantly different (Po0.001) from the normal control (9.370.11) (Fig 8, Table 2). 3.10. Viability of normal human peripheral blood mononuclear cells At 24 h of PBMNCs culture the viable cell count in control, imatinib mesylate (100 mg/ml), Ara-C (100 mg/ml) and TSE (100 mg/ml)-treated cells were (1.5870.06) 105, (1.1970.04) 105, (1.2870.04) 105 and (1.2670.04) 105, respectively. The viable cell count in Imatinib mesylate, Ara-C and TSE-treated cells decreased significantly (Po0.001) by 24.7%, 19.0% and 20.3%, respectively, as compared with control. 3.11. Cytotoxicity study of normal human peripheral blood mononuclear cells MTT assay of PBMNCs after 24 h of culture in presence and absence of Imatinib mesylate (100 mg/ml), Ara-C (100 mg/ml) and TSE (100 mg/ml)
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Fig. 5. Effect of TSE on the Annexin-V binding of U937 and K562 cells. Left panel: U937 cells (A: control, B: Ara-C standard, C: TSE-treated). Right panel: K562 cells (D: control, E: imatinib mesylate standard, F: TSE-treated). The flow cytometric analysis of percentage of Annexin-V bindings in each group are shown in the figure of respective cell groups.
revealed that the optical density of color formazan were significantly (Po0.001) reduced by 22.0%, 16.7% and 19.6%, respectively, as compared with control cells. The optical densities of color formazan in MTT assay in control, imatinib mesylate, Ara-C and TSE-treated PBMNCs were found to be 0.9970.01, 0.7770.01, 0.8270.02 and 0.7970.01, respectively. 4. Discussion It is now well established that the reduced capacity of tumor cells undergoing cell death
through apoptosis, plays a key role both in the pathogenesis and therapeutic failure of cancer. The fate of cancer cells primarily depends on the proliferation control i.e., DNA replication and apoptosis induction through the coordinated cell cycle regulation (Lam et al., 2004). In the present study, TSE showed time-dependent inhibition of U937 and K562 cell growth and proliferation. TSEinduced U937 and K562 cell growth inhibition and reduction in MTT values were correlated with the reduction in cell proliferation and cytotoxic effect of TSE. The reduction OD value has a direct
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Fig. 6. Confocal laser scanning photomicrograph of U937 and K562 cells. Upper panel: U937 (A, Control and B, TSE-treated) cells. Lower panel: K562 (C, Control and D TSE-treated) cells. * a, Marginalization of fragmented nuclei, and * b, Fragmented nuclei Magnification (1000 ).
correlation with the growth inhibitory rate and inverse relation with proliferative rate (Andrew et al., 2002). MTT is a water-soluble tetrazolium salt, which is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by mitochondrial succinate dehydrogenase. The disruption of mitochondrial dehydrogenase system due to the mitochondrial dysfunction or apoptosis, may reduce the color formazan production in the MTT assay. Cytotoxic and antiproliferative drugs may disrupt this system, which was reflected in the MTT assay. TSE suppressed the PCNA expression in U937 and K562 cells. PCNA is centrally involved in the regulation and coordination of cell cycle progression and DNA replication. It stimulates the activity of DNA polymerase d and increases its processivity by acting as a clamp platform that slides along the DNA template (Kontopidis et al., 2005). The suppression of PCNA expression as well as the cell cycle arrest primarily at G1 phase in U937
and K562 cells were correlated with the cell growth inhibition and apoptosis induction by the TSE. The antiproliferative effect of TSE and the cell cycle arrest with the simultaneous suppression of PCNA expression may explain the fact that TSE halts the cell cycle for the prevention of further cell proliferation and execution of apoptotic signaling. At 24 h, TSE-induced apoptosis in U937 and K562 cells were detected using Annexin-V-FITC binding by flow cytometry. Annexin-V binding is based on the transposition of phosphatidyl serine from the inner to the outer face of cell membrane during the early stages of apoptosis (Vermes et al., 1995). The nuclear fragmentation and formation of apoptotic bodies in U937 and K562 cells indicated the apoptogenic nature of TSE. TSE deformed the cancer cell membrane architecture and produced membrane blebbing and perforation, which is the morphological marker for apoptosis. The blebbing and pore formation may be due to the disruption of
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Fig. 7. Scanning electron photomicrograph of U937 and K562 cells. Left panel: Control (A, U937 and C, K562 ). Right panel: TSE-treated (B, U937 and D, K562) groups. * a, Membrane blebbing, * b, Membrane perforation. Magnification (3000 ).
cellular cytoskeleton element due to TSE. TSE induced DNA fragmentation was also reflected in single-cell gel electrophoresis. TSE produced significant increase in L:W in U937 and K562 cells. This is due to the movement of fragmented DNA mass of the apoptotic cells towards the anode during electrophoresis in alkaline condition. There is a direct relation of increase in L:W of DNA mass with the extent of DNA fragmentation (Singh et al., 1988). This assay is able to detect very short-lived primary DNA lesions such as single-strand break as well as double-strand breaks in the DNA of individual cells (Hartmann et al., 2004). Apoptosis leads to cytoskeletal disruption, cell shrinkage, membrane blebbing, nuclear fragmentation and disruption of DNA into fragments (Fairbairn et al., 1996). Recent observation on the studies of Chinese toad (B. b. gargarizans) skin preparation (Chan Sue) with flow cytometry, agarose gel electrophoresis of
DNA and fluorescent staining in human bladder carcinoma cell line, indicated its apoptogenic nature (Ko et al., 2005). Chan Sue induced apoptosis in human leukemic cells (U937) was also reported (Watabe et al., 1997). Genomic instability due to DNA or nuclear fragmentation usually induces the activation of p53, resulting in growth arrest or apoptosis (Dasika et al., 1999). Ablation of PCNA expression or function in cells under proliferative stimuli, appears to constitute an apoptotic trigger. The tumor-suppressor protein p21 modulates cell cycle progression by direct binding to PCNA and also by inhibiting cyclin-dependent kinases (CDKs). Arresting cells in G1, p21 can inhibit Cdc2–cyclin B complexes and proliferating cell nuclear antigen preventing interaction of the latter with other components of the DNA polymerase complex (Vidal and Koff, 2000; Waga et al., 1994). At the cellular level the competition between p21 and DNA replication factors for binding to PCNA is believed
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Fig. 8. Single cell gel electrophoresis (comet assay) of U937 and K562 cells. Left panel: control (A, U937 and C, K562). Right panel: TSE-treated (B, U937 and D, K562). * Tailed cells, Magnification (100 ).
Table 2 TSE-induced DNA fragmentation in U937 and K562 cells in comet assay Experimental group
Tailed cells (%)
Length–width ratio of DNA mass
Control U937 cells TSE treated (IC50 Dose) U937 cells Control K562 cells TSE treated (IC50 Dose) K562 cells
11.270.06 84.070.17 9.370.11 89.270.34
1.1270.048 2.2170.106 1.0770.078 2.1670.110
Values as mean7SEM. po0.001, significant.
to be the mechanism through which DNA synthesis is inhibited (Kontopidis et al., 2005). In the present study, the Annexin-V binding, DNA and nuclear fragmentation, membrane blebbing and the G1 arrest, all support the apoptogenic nature of TSE, whereas cell growth inhibition, suppression of PCNA expression and decreased absorbance of TSE-treated U937 and K562 cells in MTT assay indicated the antiproliferative and cytotoxic activity of TSE. However, TSE-induced growth inhibition and cytotoxicity in U937 and K562 cancer cells were found to be higher than that of the normal PBMNCs.
It may be concluded that, Indian TSE possesses potent antineoplastic agent, which is antiproliferative, cytotoxic and apoptogenic against human myeloid leukemic cells (U937 and K562). Isolation/purification of the active anticancer molecule from TSE is in progress. Acknowledgments The present study was partially financed by the University Grants Commission, New Delhi Government of India [Ref.: UGC/417/Major Research(Sc.)03; dt. 31.07.2003)]. Imatinib mesylate
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was a gift from Natco Pharma Ltd., Hyderabad, India.
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