Podophyllum hexandrum Fraction (REC-2006) Shows Higher Radioprotective Efficacy in the p53-Carrying Hepatoma Cell Line: A Role of Cell Cycle Regulatory Proteins
The present study was carried out to evaluate the radioprotective efficacy of Podophyllum hexandrum fraction (REC-2006) in hepatoma cell lines having different p53 statuses. Higher radioresistance was observed in the HepG2 (p53++) cell line in comparison to the Hep3B (p53--) cell line, indicating a plausible role of p53 in radioresistance. REC-2006 exhibited nearly twice the survival in p53-expressing HepG2 cells compared with p53-negative Hep3B cells. REC-2006 treatment alone induced p53 expression as compared with untreated controls. However, REC-2006 reduced p53 expression when treated 2 hours before irradiation as compared with the irradiated HepG2 controls, indicating that REC-2006 modulates the expression of p53 to mitigate its apoptotic effect. Induction of p21 in the REC-2006 + radiation treatment group downregulated the expression of cyclin E and CDK2, leading to a delay in the G1 phase of HepG2 cells, which provided time for DNA repair or related processes. However, no significant difference in CDC2 expression in both cell lines suggested that G2 phase arrest might not be the only responsible factor for REC-2006-mediated radioprotection. Significant induction of PCNA and GADD45 expression in HepG2 cells suggested that REC-2006 increased the percentage survival of HepG2 cells by increasing the span of time as well as efficacy for repair processes. In conclusion, REC-2006 modulated the expression of p53 and thereby promoted cell cycle arrest in the G1 phase, encouraging cell proliferation and DNA repair and thus providing significantly higher protection against acute g-radiation in the HepG2 cell line.
1Institute of Nuclear Medicine and Allied Sciences, Delhi, India2Jamia Hamdard, Delhi, India
Corresponding Author:Rakesh Kumar Sharma, Institute of Nuclear Medicine and Allied Sciences, Brig S. K. Mazumdar Road, Delhi 110054, IndiaEmail: [email protected]
Introduction
Ionizing radiation (IR) induces severe damage in various cel-lular compartments and results in complex biological responses leading to cell death. Several intracellular proteins work in an orchestrated manner to protect cells from genotoxic stress. IR perturbs normal cellular signaling and induces arrest in various phases of the cell cycle. Proteins involved in cell cycle check-points are known to play an important role in both cell survival and cell death.1 Progression of the cell cycle is promoted by cyclins and their respective CDKs, and defects in a cell-cycle checkpoint may lead to genomic instability and cell death.2,3
The p53 protein expression plays a pivotal role in deciding the fate of the cellular system after IR. Radiation-induced single- and double-strand breaks in DNA activate p53 protein expression in the cell.4,5 The p53 protein is present at the nexus of the web of intracellular signaling pathways that not
only coordinate cell cycle arrest and apoptosis but also play an important role in DNA repair.6-8 The p53-regulated CDK inhibitor p21waf1/cip1 is a primary mediator of p53-dependent G1 cell cycle arrest following DNA damage.8 The p21 protein negatively regulates the cell cycle progression in the G1-S phase by reducing the activity of cyclin E/CDK2 and cyclin D/CDK4 complexes.9
As long as there is no DNA damage or other stress, p53 remains latent and does not interfere with normal cellular trans-action/processes. IR-induced DNA damage accumulates and
activates p53 protein, which has a pivotal role in deciding the fate of cells by coordinating cell cycle arrest, managing apop-totic events, and modulating the DNA repair processes indirectly by inducing activation of the antioxidant enzyme glutathione peroxidase,10 activation of free radical scavenging proteins, repression of oxidating molecules like nitric oxide synthase,11 and direct DNA repair by induction of DNA repair enzymes like O6-methylguanine-DNA methyltransferase12 and O6-alkylguanine-DNA-alkyltransferase.13 It has also been shown that p53 transiently activates expression of PCNA for the purpose of limited DNA repair.
The present studies were carried out to evaluate the radio-protective efficacy of Podophyllum hexandrum fraction REC-2006 (nonpolar chloroform fraction of P hexandrum growing at an altitude of approximately 4000 m in the Himalayan region) in 2 cell lines of the same origin but with different p53 status. For this purpose, HepG2 (p53++, carry-ing wild type functional p53 gene) and Hep3B (p53--, p53 deficient) were chosen as the experimental models to evalu-ate the radioprotective efficacy of REC-2006. Our findings show that REC-2006 renders significant (P < .05) protection in the wild type functional p53 carrying cell line as com-pared with the p53 null cell line when administered 2 h before exposure to acute IR. Even though the radioprotec-tive properties of P hexandrum were investigated earlier and established in our institute,14-24 the present observations further open the window for use of REC-2006 as a radiopro-tector during planned radiation exposures (rescue operation as well as in radiotherapy of tumor cells) because it is well known that more than 60% of the tumor has cells with mutated/nonfunctional p53 protein, which is otherwise required for regulation of downstream genes responsible for cell cycle regulation and DNA repair. Our results indicate a potential for differential radioprotection of p53-expressing cells and p53-deficient cells, particularly during radiotherapy.
Materials and MethodsReagents
All reagents used were of analytical grade and standard make. Culture medium MEM, antibiotics (penicillin G and strepto-mycin), trypsin, and fetal bovine serum were procured from HiMedia, India, and Sigma Aldrich, USA. Mouse monoclonal antibodies for the detection of human p53, p21, cyclin E, CDK2, cyclin B1, CDC2, PCNA, GADD45, and b-actin pro-teins and alkaline phosphate-conjugated secondary antimouse antibodies were procured from Santa Cruz Biotech (Santa Cruz, CA). MTT, DTT, EDTA, PMSF, NP-40, BCIP-NBT reagent, and protease inhibitors were procured from Sigma Aldrich, St Louis, MO. Lysine, Tris-base and SDS were obtained from Merck, Darmstadt, Germany, and nitrocellu-lose membrane was purchased from Millipore, USA.
Plant Extract
Dried rhizomes of P hexandrum Royle (family: Berberida-ceae/Podophyllaceae) growing at an altitude of 4000 m in the Himalayan region were provided by DIHAR (Defence Insti-tute of High Altitude Research), formerly Field Research Laboratory, Leh (Jammu and Kashmir, India). The plant material was identified by Dr Rajesh Arora, Scientist, INMAS, Delhi, India, based on botanical characters. A voucher speci-men (INM-PH-FRL) has been deposited both at DIHAR, Leh, India, and INMAS, Delhi, India. The rhizomes were finely ground, and the dried powder (10 g/100 mL, w/v) of P hexandrum was transferred to a Soxhlet apparatus and extracted thrice with different solvents (1:6 ratio) of increas-ing polarity—namely, hexane, chloroform, alcohol, 50% alcohol in water, and water subsequently for over the course of 24 to 72 h. The respective filtrates were combined.14 All the extracts were filtered through Whatman filter paper No. 3 fol-lowed by filtration through a 0.22 mm filter (Millipore, USA). Extracts were then concentrated by solvent evaporation under reduced pressure in a rotary evaporator (Buchi, Flawil, Swit-zerland) and dried. Out of the 5 different extracts prepared, the dried chloroform extract (REC-2006) was used for the present study.24
Cell CultureThe human hepatoma cell lines—HepG2
(p53++,
carrying
wild type p53) and Hep3B (p53--, carrying p53 null)—were purchased from the National Centre for Cell Science, Pune, India. Cells were maintained as monolayer culture in MEM (supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, 100 mg/mL of streptomycin, pH 7.4) at 37°C in a humidified 5% CO
2 incubator (Binder, Germany) in 25 cm2
culture flasks (Nunc, USA.). Cells were subcultured twice a week. All the experiments were done when cells reached about 70% confluency.24
Harvesting of Adherent CellsCells were harvested using 0.25% trypsin in Hank’s balanced salt solution. In brief, after removing the medium, 1.0 mL of chilled trypsin (0.25% v/v) was poured into the culture flask (T25) and left for 30 s at room temperature. After decanting excess trypsin, the culture flasks were incubated at 37°C till the cells started rolling off. Harvested cells were counted, and proper dilutions were made by adding complete MEM medium to the cell suspension for further experimentation.
IrradiationA 60Co gamma chamber (Model 220, Atomic Energy of Canada Ltd) was used to deliver desired radiation doses (dose rate of 43.8 cGy/min). Cells were cultured in culture flasks (25 cm2)
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and irradiated at different doses (0.5-12 Gy). Culture flasks fitted with a filter cap (Nunc, USA) were used to avoid the gen-eration of hypoxic conditions in the irradiation chamber.
MTT AssayCell proliferation was detected by MTT (Sigma, St Louis, MO) assay. HepG2 and Hp3B cells were harvested in the log phase and were seeded on a 96-well plate (2 × 104 cells/100 mL/well) for 24 h. Cells were divided into a control (DMSO) group and a REC-2006 group. The concentration of REC-2006 added was 10 to 10-7 mg/mL of final volume. After 60 h of incubation, 10 mL MTT (5 g/L) was added and incubated at 37°C for 4 h. DMSO (75 mL) was added to each well, and the plate was oscillated for 10 min until the crystals were dissolved com-pletely. Absorbance (A) was detected with an enzyme calibrator at 560 nm. Cell viability was calculated according to the for-mula (A of study group/A of control group) × 100%. The experiment was repeated thrice.24
Colonogenic Assay for Cell SurvivalHarvested cells (HepG2 & Hep3B) were pipetted several times to make a single-cell suspension and counted. Viability of cells was tested by the dye exclusion test (0.1%trypan blue). Nearly 300 cells were seeded in 60-mm culture dishes (Tarsons, India) and incubated for 12 h. The attached cells were then treated with REC-2006 2 h before g-radiation. After various treatments, the cells were further incubated at 37°C with 5% CO2 in a humidified condition. Colonies appeared after nearly 2 weeks of incubation and were counted. All experiments were carried out in triplicate.
Experimental PlanHepG2 and Hep3B cells were divided into the following 4 groups in triplicates:
Group I: untreated control of HepG2 and Hep3B cellsGroup II: HepG2 and Hep3B cells treated with differ-
ent concentrations (1 to 10-7 mg/mL) of REC-2006Group III: HepG2 and Hep3B cells exposed to differ-
ent radiation doses (0.5-12 Gy)Group IV: HepG2 and Hep3B cells exposed to radia-
tion doses 0.5 to 2.5 h after REC-2006 (10-3 to 10-7 mg/mL) treatment
After irradiation, the culture medium (MEM) was replaced with fresh REC-2006-free complete MEM culture medium, and cells were further incubated at 37°C with 5% CO
2 in a
humidified condition for 15 days. Colonies were counted after the incubation period and percentage survival was calculated. All experiments were carried out in triplicate.
Similarly, HepG2 and Hep3B cells were divided into the above-mentioned 4 treatment groups, and after final treatment,
cells were harvested at 2, 4, 8, and 10 h for comet assay and at 8 h for protein expression studies.
DNA Fragmentation AssayTo evaluate the effect of irradiation and REC-2006 treatment on human HepG2 and Hep3B cells, single-cell gel electro-phoresis assay was performed.24 In brief, cells of different treatment groups were trypsinized, washed in PBS and mixed with prewarmed 0.75% (500-600 mL) ultra-low gelling aga-rose (Sigma Aldrich, St Louis, MO) and layered on microscope slides precoated with 0.1% normal agarose. Slides were incu-bated at 4°C to allow the formation of agarose gel. They were then submerged in the lysis buffer (2.5% sodium dodecylsul-fate, 1% sodium sarcosinate, and 25 mM EDTA, pH 9.5) for 15 min at 25°C. Slides were washed in distilled water for 5 min at 10°C. Electrophoresis was carried out in electrophore-sis buffer (90 mM Trizma base, 90 mM boric acid, and 2.5 mM EDTA, pH 8.3) at 2 V/cm for 5 min. After a brief rinse in distilled water, slides were dried at 45°C and stored in a cool humid box until use. After rehydration of slides in distilled water for 5 min, comets were stained with propidium iodide (25 mM in phosphate buffered saline) and observed under a fluorescence microscope (Olympus Optical Co, Tokyo, Japan). Analysis of comets was carried out by comet score software (TriTek Comet Score Version 1.5.0).24
Extraction of Cytoplasmic and Nuclear Protein FractionHepG2 cells were harvested by trypsinization and centrifuged at 100g for 5 min. The pellet was resuspended in 300 mL of buffer A (50 mM NaCl, 10 mM HEPES pH 8.0, 500 mM sucrose, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM sperm-ine, 0.2% triton X-100) containing b-mercaptoethanol and the protease inhibitors, that is, PMSF, leupeptin, aprotinin, and pepstatin. After 15 min of incubation on ice, the suspension was centrifuged at 1400g for 10 min. The supernatant (cyto-plasmic fraction) was collected and stored, whereas the pellet was washed with 200 mL buffer B (50 mM NaCl, 10 mM HEPES pH 8, 25% glycerol, 0.1 mM EDTA, 0.5 mM spermi-dine, 0.15 mM spermine) and then resuspended in 100 mL buffer C (350 mM NaCl, 10 mM HEPES, 25% glycerol, 0.1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine). After centrifugation at 17 000g, the supernatant (nuclear fraction) was collected and stored at 4°C.24
Protein EstimationTotal soluble protein contents in the different nuclear and cytoplasmic fractions were estimated as described earlier.17 Briefly, 10 mL of the sample was mixed with 90 mL distilled water. After thorough mixing, Bradford reagent (1.0 mL) was added, and absorbance was recorded at room temperature within 20 to 30 min at 595 nm using a microtiter plate reader
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(Wallac, USA). The amount (mg) of protein was quantified using the BSA standard curve.
SDS-PAGE Analysis of ProteinsFor SDS-PAGE analysis of the different protein fractions, 10% polyacrylamide gels of 0.75 mm thickness were prepared.18 Protein fractions were added to SDS-PAGE sample buffer (0.0625 M Tris–HCl, pH 6.8; 2% w/v SDS; 5% v/v glycerol; 2% v/v 3-mercaptoethanol; 0.01% w/v bromophenol blue) and heated in a boiling water bath for 2 to 3 min. Equal amounts of protein samples (10 mg) were loaded in each well. Electrophore-sis was carried out at a constant voltage (stacking at 60 V, resolving at 70 V). After electrophoresis, the gels were stained with gentle shaking in 0.1% Coomassie brilliant blue R-250 in methanol:glacial acetic acid:water (4:2:4, v/v/v) at room tem-perature and destained in a washing solution (methanol:acetic acid:water, 1:0.7:8.3) to obtain distinct bands over a clear back-ground. The gels were stored in 0.1% acetic acid for future analysis.18
Western Blot AnalysisFor Western blot analysis, proteins were transferred electropho-retically (100 V, 1 h) onto a nitrocellulose membrane using a mini-trans-blot assembly (Bio-Rad, Hercules, CA). The nitro-cellulose membrane was blocked in a blocking solution (containing 5% w/v skimmed milk in TBS) for 2 h at room tem-perature. The expression levels of p53, p21, cyclin E, CDK2, cyclin B1, CDC2, PCNA, GADD45, and b-actin were analyzed by probing with the respective mouse monoclonal antibodies (1:1000 dilution) having cross-reactivity with human proteins. Following 3 washes of 15 minutes each in washing buffer (TBS, 0.2% Tween 20), the membranes were incubated in TBS con-taining goat antimouse IgG alkaline phosphate conjugated secondary antibodies (1:10 000 dilution). The membranes were again washed (3 times each for 15 minutes) with washing buffer and then treated with BCIP-NBT reagent (Sigma, St Louis, MO) for 10 to 30 minutes.18 The protein bands obtained were further subjected to densitometric analysis. The quantification of individual protein bands was done using Alpha Ease FC 4.0.0 software (Alpha Innotech, India). The expression of b-actin was measured as an internal standard.
Statistical AnalysisThe data are presented as mean ± standard deviation of 3 sep-arate experiments each performed in triplicate. The statistical analysis of variance (2-way ANOVA) was performed for mul-tiple comparisons followed by a post hoc test. The effect of different concentrations on the survival of cell lines was ana-lyzed using Dunnet’s t test with 2 controls and 5 test values. Correlation analysis was performed to evaluate various dose-dependent effects. All the statistical tests were performed
using statistical software SPSS-Version-11. Significance was tested and P < .05 was considered as level of significance.
ResultsCytotoxic/Proliferative Effect of REC-2006 on HepG2 and Hep3B Cells
To evaluate the cytotoxic and cell proliferative effects of REC-2006, the MTT assay was carried out as described in the Materials and Methods section. Both HepG2 and Hep3B cell lines exhibited similar toxic and proliferative responses toward REC-2006 in a dose-dependent manner. The median lethal con-centration (LC
50) was observed to be 10-1 mg/mL for both
the cell lines. However, cell proliferative activity of REC-2006
was observed at a concentration 100 times lower than LC50
(Figure 1). The proliferative concentration range (10-3 mg/mL to 10-7 mg/mL) of REC-2006 was used for the evaluation of radio-protective efficacy in HepG2 and Hep3B cell lines.
Effect of g-Radiation on HepG2 and Hep3B Cell LinesTo determine the effect of g-radiation on HepG2 and Hep3B cells, a colony formation assay was carried out. Higher radiore-sistance was observed in the HepG2 (p53++) cell line (LD50: 7.5 Gy) as compared with the p53-deficient Hep3B (p53--) cell line (LD50: 2.2 Gy). Approximately 80% ± 3% (P < .05) mortal-ity was observed in HepG2 (p53++) cells at 10 Gy (LD
80 = 10
Gy). In contrast, a similar level (~80%) of lethality was observed in Hep3B (p53--) cells at 3.7 Gy only (LD
80 = 3.7 Gy; Figure 2).
Radiation dose modification factor (DMF) was evaluated as 1.72 ± 0.2 and 1.26 ± 0.3 at LD
50 for HepG2 and Hep3B cell
lines, respectively. Higher radioresistance observed in HepG2
Figure 1. Effect of various concentrations (mg/mL) of drug (REC-2006) on percentage growth of HepG2 and Hep3B cell lines with correlation coefficients of R2 = 0.837 and R2 = 0.840, respectively, with respect to dose of the drug. Growth response is not significantly different in both the cell lines with LC
50 = 1 mg/mL
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(p53++) cells, as compared with Hep3B (p53--) cells, indicated the plausible role of p53 in differential radiation response.
Evaluation of Radioprotective Efficacy of REC-2006 in Human Hepatoma Cell LinesTo evaluate the radioprotective efficacy of REC-2006 in HepG2 and Hep3B cell lines, varying concentrations (10-3 to 10-7 mg/mL) of REC-2006 were used. The most optimal (maxi-mum) survival (80% ± 3.2%) was observed in HepG2 (p53++) cell lines on REC-2006 (10-5 mg/mL) treatment 2 h before irra-diation (10 Gy; LD
80; 10 Gy). However, only 40% ± 3%
survival (maximum) was achieved at the same drug concentra-tion (10-5 mg/mL) at 3.7 Gy (LD
80; 3.7 Gy) in the Hep3B cell
line. Further increase or decrease in REC-2006 concentrations significantly (P < .05) reduced the survival percentage in both the cell lines (Figure 3A). To evaluate the effect of time on the radioprotective efficacy of REC-2006, all proliferative concen-tration ranges (10-3 to 10-7 mg/mL) were administered 0.5, 1, 1.5, 2, 2.5 h before acute radiation challenge (their respective LD
80) in both cell lines. Maximum survival was observed in
both cell lines on REC-2006 (10-5 mg/mL) treatment 2 h before irradiation. Further increase or decrease in time of prophylactic administration of REC-2006 prior to irradiation significantly (P < .05) reduced the radioprotective efficacy of REC-2006 in both cell lines (Figure 3B).
DNA Protection by REC-2006 Against Lethal IrradiationTo evaluate the REC-2006-mediated protection of DNA from acute irradiation, single-cell gel electrophoresis (comet assay) was carried out at 2, 4, 8, and 10 h of final treatment.
Quantitative analysis of DNA content present in the comet tail (percentage) was carried out using a comet score soft-ware (TriTek Comet Score Version 1.5.0). Nearly equal DNA damage (52% ± 2.3%) was caused in HepG2 and Hep3B cells when exposed to respective acute radiation doses (10 Gy for HepG2 [LD
80 = 10 Gy] and 3.7 Gy for
Hep3B [LD80
= 3.7 Gy]). Considerable differences in the
Figure 2. Effect of various doses of g radiation (0.5-12 Gy) on percentage survival of HepG2 and Hep3B cell lines with correlation coefficients of R2 = 0.946 and R2 = 0.989, respectively, with respect to radiation dose. The asterisk indicates significant difference in percentage survival of cell lines. LD
80 (acute radiation
stress) was found to be 10 Gy and 3.7 Gy, respectively (used in subsequent experiments)
Figure 3. A, Effect of various concentrations (10-3 to 10-7 mg/mL) of drug (REC-2006) on percentage survival of HepG2 and Hep3B cell lines. A single asterisk indicates maximal survival in the HepG2 cell line, whereas the double asterisk indicates maximal survival in the Hep3B cell line at an optimal dose of 10-5 mg/mL with respect to untreated and radiation (LD
80 for respective cell lines) controls;
this was evaluated using Dunnet’s t test with 2 controls and 5 sample values. Significantly (P < .05) higher level of percentage survival was observed in the HepG2 cell line as compared with the Hep3B cell lines with respect to their respective LD
80 radiation
dosages. B. Effect of various treatment times (0.5-2.5 h) at optimal concentration of the drug (REC-2006), that is, 10-5 mg/mL, prior to irradiation (LD
80 radiation dosages with respect to the respective
cell lines) on percentage survival of HepG2 and Hep3B cell lines. Asterisk indicates maximal survival in the HepG2 cell line, whereas the double asterisk indicates maximal survival in the Hep3B cell line at the respective treatment times
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DNA of comet tail (in percentage) were observed in REC-2006 (2 h + radiation) and radiation-only treatment groups in both HepG2 and Hep3B cell lines at 4 h; the differences increased up to 8 h (Figure 4), but after that no discernible changes in the differences of the DNA of comet tail (per-centage) of radiation-only and REC-2006 (2 h + radiation) treatment groups were observed. These observations indi-cated that DNA protective efficacy of REC-2006 decreased 8 h after final treatment. A higher quantity (34.3% ± 2.1%) of DNA in the comet tail was observed at 8 h in Hep3B cells treated with REC-2006 2 h before irradiation, as compared with quantity of DNA present in the respective treatment group of HepG2 cells (Figure 4). As maximum DNA protec-tion was observed in both cell lines at 8 h, cellular proteins were isolated 8 h after the final treatment for immunoblot analysis of proteins related to cell cycle regulation.
Influence of REC-2006 and g-Radiation on Proteins Related to Cell Cycle RegulationTo evaluate the effect of REC-2006 and g-radiation on pro-teins related to cell cycle regulation, the expression of p53, p21, cyclin E, CDK2, cyclin B1, CDC2, PCNA, and GADD45 were carried out by immunoblot method as described in the Materials and Methods section. The expression of b-actin was evaluated for an internal standard a and it was found to be the same for both HepG2 and Hep3B cell lines. A significant (P < .05) increase in the expression of p53 was observed in HepG2 (p53++) cells treated with REC-2006 alone (30% ± 3.7%) and irradiation (73% ± 4.2%) as compared with the untreated con-trol. However, REC-2006 treatment 2 h before irradiation
decreased (16% ± 2.75%) the expression of p53 as compared with the irradiated-only group of the HepG2 cell line (Figure 5A; lanes 3 and 4). As predicted, no expression of p53 was observed in any treatment group of the Hep3B (p53--) cell line (Figure 5A).
REC-2006 treatment alone increased (30% ± 2.8%) the expression of p21waf-1/cip-1 as compared with untreated controls of the HepG2 cell line (Figure 5B; lanes 1 and 2). Although g-radiation alone induced the expression of p21 as compared with untreated HepG2 cells, REC-2006 treatment further increased (13% ± 2.3%) the expression of p21 when treated 2 h before irradiation as compared with the only-irradiated HepG2 controls (Figure 5B; lanes 3 and 4). However, no expression of p21 was observed in the untreated controls, REC-2006, and REC-2006 + radiation treatment groups of Hep3B cells (Figure 5B; lanes 1, 2, and 4). The expression of p21 was observed only in the radiation treatment group of Hep3B cells (Figure 5B; lane 3).
REC-2006 treatment alone decreased the expression of cyclin E by 14% ± 3.6% as compared with the untreated con-trol of HepG2 cells (Figure 5C; lanes 1 and 2). A significant reduction (19% ± 3.7%) in the expression of cyclin E was observed in HepG2 cells treated with REC-2006 2 h before irradiation as compared with the irradiated-only HepG2 con-trols (Figure 5C; lanes 3 and 4), whereas no such decrease was observed when REC-2006 was administered 2 h before irradiation in Hep3B cells (Figure 5C; lanes 3 and 4). Furthermore, no change in the expression of cyclin E was observed in any treatment group of the Hep3B cell line (Figure 5C; lanes 1-4). REC-2006 treatment alone reduced the expression of CDK2 (9% ± 2.7%) in HepG2 cells but failed to do the same in the Hep3B cell line (Figure 5D; lanes
Figure 4. Effect of optimal concentration of drug (REC-2006), that is, 10-5 mg/mL, given prior to irradiation on percentage DNA content in comet tail. The lower the percentage of DNA in the comet tail, the higher will be the percentage DNA repair against acute radiation stress (LD
80 = 10 Gy and 3.7 Gy with respect to HepG2 and Hep3B cell lines). Time-dependent significant (P < .05) change in percentage DNA
repair was observed till 8 h in both the cell lines with correlation coefficient of R2 = 0.8782 and R2= 0.902 in HepG2 and Hep3B cell lines, respectively. Asterisk indicates significant percentage DNA repair in HepG2 cells as compared with Hep3B cells at 4, 8, and 10 h
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Figure 5. Effect of optimal concentration of drug (REC-2006), that is, 10-5 mg/mL, given 2 h prior to irradiation (LD80 of respective cell lines) on the expression of (A) p53, (B) p21, (C) cyclin E, (D) CDK2, (E) cyclinB1, (F) CDC2, (G) PCNA, and (H) GADD 45 in HepG2 and Hep3B cell lines. Asterisk indicates significant radiomodulatory activity (increase/decrease in expression) in HepG2cell lines, whereas the double asterisk indicates the corresponding effect in Hep3B cell lines with respect to radiation (LD
80) controls
1 and 2). The expression of CDK2 was reduced (10% ± 2.7%) further on REC-2006 treatment 2 h before irradiation as com-pared with the irradiated HepG2 controls (Figure 5D; lanes 3 and 4). The expression of CDK2 was found to be much higher (41% ± 4.2%) in the REC-2006 + radiation treatment group of HepG2 cells as compared with the respective treatment group of Hep3B cells (Figure 5D; lane 4). Again, no signifi-cant change in the expression of CDK2 was observed with any treatment groups of Hep3B cells (Figure 5D; lanes 1-4).
REC-2006 treatment alone reduced the expression of cyclin B1 (12% ± 2.3%) in HepG2 cells; however, only a 5% ± 1.6% decrease was observed in the corresponding treatment group of Hep3B cells (Figure 5E; lanes 1 and 2). A significant (P < .05) decrease (20% ± 3.6%) in the expression of cyclin B1 was observed when REC-2006 was treated 2 h before irradiation as compared with the irradiated-only HepG2 controls (Figure 5E; lanes 3 and 4). However, on the other hand, a slight decrease (by 7% ± 2.6%) was observed in the corresponding treatment groups of Hep3B cells (Figure 5E; lanes 3 and 4). Although REC-2006 alone and g-radiation alone reduced the expression of CDC2 in both HepG2 and Hep3B cell lines, REC-2006 treatment 2 h before irradiation did not further reduce the expression of CDC2 in both cell lines (Figure 5F; lanes 3 and 4). Furthermore, no discernible difference in the expression pattern of CDC2 was observed in any treatment groups of HepG2 and Hep3B cell lines (Figure 5F; lanes 1-4).
An induction (46% ± 3.1%) in expression of PCNA was observed in the HepG2 cells on REC-2006 treatment 2 h before irradiation as compared with the irradiated HepG2 controls (Figure 5G; lanes 3 and 4). Although a 19% ± 2.5% increase in the expression of PCNA was observed in Hep3B (p53--) cells on REC-2006 treatment 2 h before irradiation as compared with the irradiated Hep3B control (Figure 5G, lanes 3 and 4), no
significant change (P < .05) in the PCNA expression was observed in REC-2006 2 h before the irradiated group as com-pared with the only-REC-2006 treated group of the Hep3B cell line (Figure 5G; lanes 2 and 4). A significant (P < .05) increase (by 25% ± 3.2%) in the expression of GADD45 was observed in HepG2 cells when REC-2006 was administered alone; how-ever, REC-2006 treatment alone failed to induce the expression of GADD45 in Hep3B cells (Figure 5H; lane 2). REC-2006 treatment 2 h before irradiation increased the expression of GADD45 (by 39% ± 2.4%) as compared with the irradiated HepG2 controls (Figure 5H; lanes 3 and 4). Only a 14% ± 2.6% increase was observed in the respective treatment group of the Hep3B cell line.
DiscussionP hexandrum, a high altitude Himalayan medicinal plant has been used since ancient times in the Indian and Chinese systems of medicine for the treatment of various diseases and disorders.25 However, recently, the radioprotective properties of P hexan-drum have been discovered and established in our institute.14-24 In the present study, a phytochemically characterized fraction of P hexandrum (REC-2006), containing podophyllotoxin and its derivatives (4′-demethyl podophyllotoxin-b-D glucopyranoside etc)19 was used to evaluate the radioprotective efficacy in hepa-toma cell lines having different p53 status. Observations of the present study indicated that the cytotoxic and proliferative effect of REC-2006 is independent of the p53 status of the cell lines as both p53-positive and p53-negative cell lines exhibit the median lethal concentration (LC
50) at 0.1 mg/mL (Figure 1). Higher
radioresistance as observed in HepG2 (p53++) cells (LD50
; 7.5 Gy) as compared with Hep3B (p53--) cells (LD
50; 2.2 Gy) pre-
liminarily indicate the role of p53 in radioresistance.
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Varied concentration ranges (10-3 to 10-7 mg/mL) of REC-2006 and treatment times (0.5, 1, 1.5, 2, and 2.5 h) were used to achieve maximum radioprotective efficacy in both the cell lines. Maximum radiation protection was achieved at the same concentration (10-5 mg/mL; Figure 3A) when treated 2 h before irradiation (Figure 3B) both in HepG2 and Hep3B cell lines. However, higher protection was observed in the HepG2 (p53++) cell line (80% ± 3.2%) in comparison with the Hep3B (p53--) cell line (40% ± 3%) when REC-2006 was treated 2 h before irradiation (their respective LD
80), suggesting a domi-
nant role of p53 protein expression in radioprotection offered by P hexandrum fraction REC-2006 containing podophyllo-toxin and its derivatives. This is in line with our earlier studies where lignans (podophyllotoxin and its derivatives) have been reported to contribute to radiation protection.24
The role of p53 in the activation of the antioxidant enzyme glutathione peroxidase,10 activation of free radical scaveng-ing proteins, repression of oxidating molecules like nitric oxide synthase,11 and direct DNA repair by induction of DNA repair enzymes like O6-methylguanine-DNA methyltransfer-ase12 further provides support for the idea of involvement of p53 protein in REC-2006-mediated radiation protection.
Regulation of the cell cycle during various phases follow-ing irradiation determines whether a cell will survive or not. Therefore, the role of REC-2006 in the modulation of pro-teins (p21, cyclin E, CDK2, cyclin B1, CDC2, PCNA, and GADD45) directly or indirectly involved in the regulation of the cell cycle and their implications in radioprotection were studied using HepG2 (p53++) and Hep3B (p53--) cell lines.
The cellular response to genotoxic agents like g-radiation includes an increase in the level and activity of p53.26,27 On acti-vation, p53 inhibits replication of the genome under unfavorable conditions by regulating cell cycle progression and cell viabil-ity, thereby preventing proliferation of cells with damaged genes. The high incidence of p53 mutations in human tumors suggests that these activities are central to tumor suppression. The functions of p53 largely depend on its ability to both acti-vate and repress transcription.2-27 REC-2006 treatment, before irradiation, increased the expression of p21, a downstream regu-latory protein, in the HepG2 cell line leading to G1 growth arrest. As no expression of p21 was observed in p53-null Hep3B cells in the REC-2006 + radiation treatment group, DNA damage must have been incorporated in the next generation, leading to cell death. Among p53-inducible genes, the p21WAF1/cip1 gene encodes a protein that inhibits CDK activity and leads to G1 growth arrest.9,28,29
A sharp decrease in cyclin E and CDK2 expression on REC-2006 treatment (+/- irradiation) as observed in p53-expressing HepG2 cells but not in Hep3B (p53--) cells (Figures 5C and 5D; lane 4) further supported the p21-medi-ated G1 arrest in HepG2 cells, which might have provided a sufficient span of time to repair the damage leading to radia-tion protection. Because p53 plays a significant role in cell cycle regulation after DNA damage, it can be suggested that induced expression of p53 in HepG2 (p53++) cells on
REC-2006 (+/- irradiation) treatment might have further induced the expression of p21waf/cip1, which negatively regu-lated the cyclin-E–CDK2 complex formation and thus delayed the cell cycle progression in the G1/S phase, allowing time for repair processes in HepG2 (p53++) cells. The p53-dependent activation of p21waf/cip1 and downregulation of cell cycle progression in the G1/S phase by the best-known radio-protector phosphoaminothiol WR1065 (the active metabolite of WR-2721) has been reported earlier,30 providing strong support for the present investigation. In contrast, because of the lack of p53, the expressions of cyclin E and CDK2 were found unchanged in the Hep3B (p53-) cell line on REC-2006 (+/- irradiation) treatment (Figures 5C and 5D), which might have promoted the entry of cells that were not repaired into the S phase leading to cell death. These observations were further supported by the results of comet assay (Figure 4), which show higher DNA damage in the Hep3B cell line. REC-2006-mediated induction of p53 might also have increased the expression of a novel p53 target gene desig-nated p53-inducible cell survival factor (p53CSV), an important player in p53-mediated cell survival,30 which can modulate apoptotic pathways through interaction with Hsp70, which probably inhibits the activity of Apaf-1 and release of cytochrome c from mitochondria. However, further analysis of p53CSV, Hsp70, Apaf-1, and cytochrome c is required to verify such observations.
The comparatively lower expression in cyclin B1 as observed on REC-2006 treatment 2 h before radiation in the HepG2 cell line as compared with the same treatment group of the Hep3B cell line suggested the G2 phase arrest in the HepG2 cell line. However, no significant (P < .05) difference in the expression of CDC2 in the REC-2006 + radiation treat-ment group and no significant (P < .05) difference in the expression of CDC2 in radiation-only and REC-2006 + radia-tion treatment groups of both p53-positive and p53-negative cell lines as observed in HepG2 and Hep3B cell line (Figure 5E, 5F) suggested that an arrest in the cell cycle at the G2 phase might not be the deciding factor in providing higher radioprotection in the HepG2 cell line.
To further analyze the influence of REC-2006 on cell cycle regulatory and DNA repair proteins, the expressions of PCNA and GADD45 were analyzed. An induction in PCNA and GADD45 expressions was observed in the HepG2 (p53++) cell line in the REC-2006 + radiation treatment group as compared with the irradiated control (Figure 5G, 5H), suggesting a piv-otal role of REC-2006 not only in the induction of cell cycle arrest but also in the induction of DNA repair processes. PCNA participates in both nucleotide excision repair and mismatch repair.31,32 PCNA also directly binds 2 p53-inducible proteins, GADD45 and p21,32-34 and these interactions may regulate PCNA-dependent DNA replication.35,36 These observations indicate that PCNA may integrate the cellular processes that regulate DNA replication and repair. The expression of PCNA in response to REC-2006 and g-radiation is consistent with this view. Initial investigations of PCNA regulation by p53
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demonstrated that p53, when expressed in transient cotransfec-tion experiments,37 had no effect or repressed expression from the PCNA promoter. Later observations demonstrated that wild-type p53 binds the human PCNA promoter and transactivates PCNA promoter-directed gene expression in a concentration-dependent manner; lower levels activate, whereas higher levels do not.38,39 We earlier reported that REC-2006 maintained the threshold level of p53 by increasing the expression of MDM2.24 Decrease in the expression of p53 in REC-2006 + radiation treatment group as compared with the irradiated-only HepG2 control is consistent with the earlier finding. These observations suggested that even if REC-2006 treatment alone induced the p53 expression in the HepG2 cell line, REC-2006 downregulated the expression of p53 when treated 2 h before irradiation to bring the p53 expression within the threshold level to optimally activate the expression of PCNA for DNA repair and survival. The p53-mediated regula-tion of PCNA expression in cells exposed to IR has been reported.40 Binding of p21 with the C-terminal domain of PCNA leads to the loss of its ability to activate DNA poly-merase-d and thus inhibits DNA replication, which further leads to cell cycle arrest at the G1 phase.35 Induction of p21 in the REC-2006 + radiation treatment group of the HepG2 cell line supported these observations. Furthermore, p21 forms a quaternary complex with cyclins, CDKs, and PCNA and acts to regulate cyclin/CDK activity to directly affect DNA replication through its interaction with PCNA.41 The role of PCNA and GADD45 in nucleotide excision and single-strand DNA break repair,35,42 provided support to higher radiation protection ren-dered by REC-2006 in the HepG2 cell line. Lower expression of GADD45 in p53-deficient Hep3B cells on REC-2006 treat-ment suggested that REC-2006 might not be capable of inducing GADD45-mediated cell cycle arrest and ssDNA repair in the absence of p53 expression and, thus, will be unable to protect Hep3B (p53--) cells from radiation-induced cell death. Although lower radiation protection (percentage) was observed in the Hep3B cell line as compared with the protec-tion rendered in the HepG2 cell line, the radiation protection observed in the Hep3B cell line can be explained by the proper-ties of P hexandrum, which offers radioprotection by its antioxidant activity, free-radical scavenging activity, antilipid peroxidation activity, and stabilization of mitochondrial mem-brane potential.15,16 However, all these properties of P hexandrum must have protected both the cell lines irrespective of p53 status. As tumor and normal cells generally have differ-ent p53 statuses, REC-2006 treatment before radiation therapy can be promising; however, the role of REC-2006 in the modu-lation of proapoptotic and prosurvival proteins needs further analysis.
ConclusionFrom the observations of the present study, it can be concluded that p53 expression plays a significant role in the radiation protection offered by REC-2006 and that a threshold level of
p53 is required to perform its radioprotective function. REC-2006-induced p53 expression results in a delay in the cell cycle at the G1 phase, which provides sufficient time for the cells to undergo repair. The p53 protein expression further induces cell proliferation and inhibits cell death, thereby pro-viding protection against lethal irradiation in HepG2 (p53++) cells. In contrast, the absence of p53-mediated cell cycle regu-lation in Hep3B (p53--) cells makes them highly radiosensitive because of their inability to repair radiation-induced damage. In conclusion, REC-2006 is able to provide radioprotection to cells carrying wild type p53 selectively, suggesting that P hexandrum has potential for use in radioprotection during planned radiation exposures, for example, rescue missions, space missions, and radiotherapy, where it holds immense promise for providing differential radioprotection.
Abbreviations
CDK cyclin-dependent kinasePCNA proliferating cell nuclear antigenGADD45 growth arrest DNA damage-45MTT 3 - ( 4 - , 5 - d i m e t h y l t h i a z o l - 2 - y l )
The authors would like to express their gratitude to the Director, INMAS, for providing necessary facilities.
Declaration of Conflicting Interests
The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.
Funding
This work was sponsored by Defence Research and Development Organization (DRDO), Ministry of Defence, Government of India. Dr Pankaj Kumar Singh received financial support in the form of a research fellowship by the Indian Council of Medical Research (ICMR), New Delhi, India.
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