Activation of Caspases and Poly (ADP-Ribose) Polymerase Cleavage to Induce Apoptosis in Leukemia HL-60 Cells by Inula racemosa
Keywords : Inula racemosa, Compositae, Cytotoxicity, Tumor growth inhibition, Apoptosis
Abstract
Inula racemosa Hook.f., commonly known as Pushkarmula (Compositae), has been used as a traditional drug in India, China, and Europe. In the present study, 95% ethanolic extract of roots and its fractions (n-hexane, chloroform, n-butanol, and aqueous) were evaluated for in vitro cytotoxicity against cancer cell lines of colon, ovary, prostate, lung, CNS, and leukemia. The n-hexane fraction containing alantolactone and isoalantolactone as its major constituents was further studied for its mode of action in HL-60 cells. The lowest IC₅₀ value of n-hexane fraction was 10.25 μg/ml for Colo-205, a colon cancer cell line, whereas 17.86 μg/ml was the highest IC₅₀ value observed against CNS cancer cell line SF-295. Further studies on HL-60 cells treated with n-hexane fraction at 10, 25, and 50 μg/ml for 6 h revealed that it induces apoptosis through intrinsic as well as extrinsic pathways by generating reactive oxygen species (ROS) intermediates. Mitochondrial dysfunction prompted the release of cytochrome c, translocation of pro-apoptotic protein (Bax), activation of caspase cascade, resulting in the cleavage of specific substrates for caspase-3 such as poly (ADP-ribose) polymerase (PARP), which eventually leads to apoptosis. The results of present study strongly support further research and development of bioactive constituents from Inula racemosa as potential anticancer agents with possible therapeutic implication.
1. Introduction
Drug discovery from medicinal plants has played an important role in the treatment of cancer over the last half century. Herbal medicines, botanicals, dietary supplements, and edible plants have been used in cancer chemoprevention and treatment. Inula racemosa Hook.f., commonly known as Pushkarmula (Compositae), is a well-documented Indian medicinal plant. The plant has been used as a traditional drug in India, China, and Europe. Plant extract is used for abdominal pain, acute enteritis, and bacillary dysentery. Preparations of its roots are used in indigenous medicine as expectorant, tonic, and in folk medicine of several ethnic groups against a variety of ailments including asthma, cough, bronchitis, lung disorders, indigestion, chronic enterogastritis, infectious and helminthic diseases. In addition, roots of the plant are widely used in the treatment of cardiovascular diseases and tuberculosis.
Inula racemosa produces a wide array of sesquiterpenoid, especially sesquiterpene lactones including eudesmanolides, guaianolides, and germacranolides as their main secondary metabolites and essential oils. The phytochemical investigation of the plant showed the presence of alantolactone, isoalantolactone, dihydroalantolactone, dihydroisoalantolactone, sitisterol, daucosterol, inunolide, aplotaxene, phenyllacetonitile, and isoinunal. Two major constituents, alantolactone and isoalantolactone, possess anti-fungal and anthelmintic activities. The anti-ulcer drug Alanton, consisting of a mixture of the alantolactones, is well known to industry. Moreover, sesquiterpene lactones also possess a wide spectrum of biological activities including anti-inflammatory, fungicidal, and anticancer properties.
In the present study, 95% ethanolic extract of Inula racemosa roots and its four fractions (n-hexane, chloroform, n-butanol, and aqueous) were evaluated for their in vitro cytotoxic potential against human cancer cell lines of various tissues such as colon, ovary, prostate, lung, CNS, and leukemia. Further studies on n-hexane fraction treated HL-60 cells revealed that it induces apoptosis through intrinsic (mitochondria-mediated) as well as extrinsic (death receptor-mediated) pathways by generating reactive oxygen species (ROS) intermediates. Mitochondrial dysfunction prompted the release of cytochrome c, translocation of pro-apoptotic protein (Bax), activation of caspase cascade, resulting in the cleavage of specific substrates for caspase-3 such as poly (ADP-ribose) polymerase (PARP), which eventually leads to apoptosis.
2. Materials and Methods
2.1. Chemicals and Antibodies
RPMI-1640 medium, DCFH-DA, Rhodamine-123, propidium iodide (PI), sulphorhodamine B (SRB), dimethyl sulphoxide (DMSO), DNase-free RNase, proteinase-K, PMSF, protease inhibitor cocktail, triton-X100, penicillin, streptomycin, L-glutamine, pyruvic acid, camptothecin, fetal bovine serum, and AnnexinV-FITC apoptosis detection kit were purchased from Sigma Aldrich, USA. Other reagents and antibodies were sourced as described in the original article.
2.2. Extraction, Fractionation, and Isolation
Roots of Inula racemosa were collected from Jammu and Kashmir, India, identified, and authenticated. Dried and powdered roots were extracted with 95% ethanol, and the extract was fractionated sequentially with n-hexane, chloroform, n-butanol, and water. The n-hexane fraction was further processed to isolate alantolactone and isoalantolactone, confirmed by spectroscopic methods.
2.3. Preparation of Standards and Sample
Standards of alantolactone and isoalantolactone were prepared in methanol. The n-hexane fraction was dissolved in methanol and filtered before HPLC analysis.
2.4. LC-MS Analysis
LC separations were performed using an RP-18 column with a water:acetonitrile mobile phase. The n-hexane fraction was found to contain 19.28% alantolactone and 37.06% isoalantolactone.
2.5. Cell Culture and Treatment
Human cancer cell lines (colon, ovary, prostate, lung, CNS, and HL-60 leukemia) were cultured in RPMI-1640 medium. Human PBMCs were isolated from blood. Extracts and fractions were prepared and diluted in culture medium for cytotoxicity assays.
2.6. In Vitro Cytotoxicity
Cytotoxicity was assessed using the SRB assay for adherent cells and MTT assay for HL-60 and PBMCs. IC₅₀ values were determined by regression analysis.
2.7. Annexin V/PI Flow Cytometric Analysis
Phosphatidylserine exposure was measured in HL-60 cells treated with n-hexane fraction using Annexin V-FITC/PI staining and flow cytometry.
2.8. DNA Agarose Gel Electrophoresis
DNA fragmentation was analyzed in treated HL-60 cells to assess apoptosis.
2.9. DNA Content and Cell Cycle Phase Distribution
Cell cycle analysis was performed by PI staining and flow cytometry to determine the sub-G₀ apoptotic fraction.
2.10. Mitochondrial Membrane Potential
Changes in mitochondrial membrane potential (ΔΨmt) were measured using Rhodamine-123 staining and flow cytometry.
2.11. Measurement of Intracellular ROS
ROS levels were assessed using DCFH-DA staining and flow cytometry.
2.12. Caspase Assays
Activities of caspase-3, -6, -8, and -9 were measured colorimetrically in cell lysates.
2.13. PARP Cleavage
Cleavage of PARP was analyzed by flow cytometry using a specific antibody.
2.14. Preparation of Lysates and Western Blot
Cytosolic, mitochondrial, and whole cell lysates were prepared for immunoblotting to detect cytochrome c, Bax, and TNF-R1.
2.15. Statistical Analysis
Data are expressed as mean ± SE. Statistical significance was determined by Student’s t-test.
3. Results
3.1. In Vitro Cytotoxicity
The n-hexane fraction exhibited maximum cytotoxicity among all fractions, with the lowest IC₅₀ value of 10.25 μg/ml for Colo-205 cells and the highest (least potent) IC₅₀ of 17.86 μg/ml for SF-295 CNS cells. The n-butanol and aqueous fractions did not show significant cytotoxicity. PBMCs remained viable under similar conditions.
3.2. Apoptosis Induction by Annexin V/PI Binding
HL-60 cells treated with n-hexane fraction showed a concentration-dependent increase in early apoptotic (Annexin V positive) and late apoptotic/necrotic cell populations, confirming induction of apoptosis.
3.3. DNA Fragmentation
A distinct DNA laddering pattern was observed in HL-60 cells treated with n-hexane fraction, indicating apoptosis. No such fragmentation was observed in treated PBMCs.
3.4. Cell Cycle Analysis
Treatment with n-hexane fraction led to a concentration-dependent increase in the sub-G₀ (apoptotic) fraction in HL-60 cells, indicating cell cycle arrest and apoptosis.
3.5. Mitochondrial Membrane Potential
A significant, concentration-dependent loss of mitochondrial membrane potential was observed in HL-60 cells treated with n-hexane fraction.
3.6. ROS Generation
Treatment with n-hexane fraction induced a concentration-dependent increase in ROS levels in HL-60 cells.
3.7. Caspase Activation
The n-hexane fraction induced significant, concentration-dependent increases in the activities of caspase-3 (~5-fold), caspase-6 (~4-fold), caspase-8 (~2.5-fold), and caspase-9 (~4-fold) in HL-60 cells.
3.8. PARP Cleavage
A concentration-dependent increase in PARP cleavage was observed in HL-60 cells treated with n-hexane fraction, confirming activation of the caspase cascade.
3.9. Cytochrome c Release and Bax Translocation
Immunoblot analysis showed significant increases in cytochrome c release into the cytosol and Bax translocation to mitochondria in a concentration-dependent manner.
3.10. TNF-R1 Expression
Treatment with n-hexane fraction caused a significant, concentration-dependent increase in TNF-R1 expression, suggesting involvement of the extrinsic apoptotic pathway.
4. Discussion
The present study demonstrates that the n-hexane fraction of 95% ethanolic extract from Inula racemosa roots exhibits in vitro cytotoxicity against various human cancer cell lines and induces apoptosis in HL-60 leukemia cells. Apoptosis was evidenced by Annexin V binding, DNA fragmentation, increased sub-G₀ DNA fraction, and morphological changes. The induction of apoptosis involved both intrinsic (mitochondrial) and extrinsic (death receptor) pathways, as indicated by loss of mitochondrial membrane potential, ROS generation, cytochrome c release, Bax translocation, caspase activation, PARP cleavage, and increased TNF-R1 expression.
The results suggest that apoptosis is mediated by activation of caspases, which degrade PARP and other proteins, leading to the characteristic features of apoptosis. Mitochondrial dysfunction, ROS production, and release of cytochrome c are central to the intrinsic pathway, while TNF-R1 upregulation and caspase-8 activation indicate involvement of the extrinsic pathway.
Importantly, the n-hexane fraction did not exhibit cytotoxicity toward normal PBMCs, highlighting its potential as a selective anticancer agent. Further phytochemical investigation is warranted to identify the active principles responsible for these effects.