A traditional poly herbal medicine “Le Pana Guliya” induces apoptosis in HepG2 and HeLa cells but not in CC1 cells: an in vitro assessment
© The Author(s) 2017
Received: 8 September 2016
Accepted: 20 December 2016
Published: 3 January 2017
“Le Pana Guliya” (LPG) is a polyherbal formulation which is used to treat different types of cancers in traditional medicine. In this study we describe in vitro efficacy and mechanism of action of LPG on two cancer cell lines (HepG2 and HeLa) compared with a normal cell line CC1. The MTT, LDH assays and protein synthesis were used to study antiproliferative activity of LPG while NO synthesis and GSH content were assayed to determine the oxidative stress exerted by LPG. Rhodamine 123 staining, caspase 3 activity, DNA fragmentation and microscopic examination of cells stained with ethidium bromide/acridine orange were used to identify the apoptosis mechanisms associated with LPG. The LPG showed the most potent antiproliferative effect against the proliferation of HepG2 and HeLa cells with an EC50 value of 2.72 ± 1.36 and 19.03 ± 2.63 µg/mL for MTT assay after 24 h treatment respectively. In contrast, CC1 cells showed an EC50 value of 213.07 ± 7.71 µg/mL. Similar results were observed for LDH release. A dose dependent decrease in protein synthesis was shown in both cancer cell types compared to CC1 cells. The reduction of GSH content and elevation of cell survival with exogenous GSH prove that the LPG act via induction of oxidative stress. LPG also stimulates the production of NO and mediates oxidative stress. Rhodamine 123 assay shows the mitochondrial involvement in cell death by depletion of Δψ inducing downstream events in apoptosis. This results in increase in caspase-3 activity eventually DNA fragmentation and LPG induced apoptotic cell death. In conclusion the present study suggested that the LPG exerted an anticancer activity via oxidative stress dependent apoptosis. Therefore present study provides the scientific proof of the traditional knowledge in using LPG as an anticancer agent.
KeywordsAnti-cancer activity MTT assay LDH assay GSH Rhodamine123 Cytotoxicity
Plants, marine, and micro-organisms are rich sources of diverse and complex compounds; many of which have potent biological activities that may be beneficial in treating human disease. Early civilizations realized the healing potential of natural products, especially those found in plants. The “Ebers Papyrus”, written in 1500 B.C, outlined the Egyptians usage of 700 drugs, most of which were derived from plants . Over the past two centuries, scientists have employed varying methods of extraction to isolate and identify the “active” compounds of these natural remedies and in doing so uncovered a wealth of chemical diversity. Cancer is characterized by uncontrolled cell division. Almost all cell types can initiate cancerous growth; as such more than 100 malignancies have been recognized . Our understanding on methods of treatment and diagnosis of these diseases has made great strides in the last 50 years in terms of mortality and morbidity; however, many forms of cancers still lack effective treatment options. The ineffectiveness of current chemotherapeutic agents warrants investigations into alternative compounds to improve today’s therapy regimens or to act as a means of chemoprevention. In effort to develop therapeutics for cancer and other diseases, pharmaceutical companies often screen large chemical libraries for potential leads. While screening of these libraries can identify potential leads, compounds synthesized by natural sources also have potential in cancer treatment .
The emphasis placed on development of natural products or analogues thereof as therapeutics has proven beneficial. Bark from the Pacific Yew tree (Taxus brevifolia), found in the Northwest United States, yields Paclitaxel (Taxol®) which is used clinically to treat Kaposi sarcoma, breast, non-small cell lung, and ovarian cancer [4, 5]. In addition, an analogue of paclitaxel, docetaxel (Taxotere®), has been developed to treat breast, gastric, prostate, and head and neck cancers . Traditional and indigenous practitioners in Sri Lanka have been treating cancer patients using plant based formulations. In addition to use of a single plant, poly herbal formulations of drugs are intensively used in Sri Lanka. The poly herbal drug named “Le Pana Guliya” (LPG) is a well known drug among the traditional medicinal practitioners which is used to treat various types of cancers. The protocol and the method of preparation are recorded in ‘Ola leaf inscriptions’ belong to their families and passing from one generation to the next.
The mechanism of action of poly herbal drug of this nature with large number of different plant components cannot be revealed through conventional bioassay-guided fractionation. Keeping above in view, the present study was aimed at investigating the cytotoxicity effect of a poly herbal drug “Le Pana Guliya (LPG)” against two different of cancer cell lines compared to the normal healthy cells and reveals the mechanism of action of its cytotoxicity.
Chemicals and equipment
Chemicals needed for cell culture, Folin-Ciocalteu reagent, sodium carbonate (Na2CO3), aluminum chloride (AlCl3), sodium nitrite (NaNO2), sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St Louis, MO63178, USA). TritonX-100 was purchased from Fluka. Tris base was purchased from Promega (Madison, WI 53711–5399, USA). Other chemicals were obtained from Sigma-Aldrich Co (St Louis, MO, USA) unless indicated otherwise. All chemicals used were of analytical grade.
Shimadzu UV 1601 UV visible spectrophotometer (Kyoto, Japan) was used to measure the absorbance. LFT 600 EC freeze dryer was used to obtain the freeze dried powder of the poly herbal drug. Cells were incubated at 37 °C in a humidified CO2 incubator (SHEL LAB/Sheldon manufacturing Inc. Cornelius, OR 97113, USA). Inverted fluorescence microscope (Olympus Optical Co. Ltd. 1X70-S1F2, Japan) for observation of cells, and photographs were taken using microscope digital camera (MDC200 2 M PIXELS, 2.0 USB). Deionized water from UV ultra-filtered water system (Waterproplus LABCONCO Corporation, Kansas city, Missouri 64132–2696) and distilled water was used in all experiments.
Human hepatocellular carcinoma cell line (HepG2) and human cervical adenocarcinoma cell line (HeLa) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% heat inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL). The cells were maintained in 25 cm2 plastic tissue culture flasks at 37 °C in a humidified atmosphere containing 5% CO2 in air. Exponentially growing cells were used in all experiments. The normal rat fibroblast (CC1) cell line was employed as the control. In all experiments cells were suspended in the growth medium and seeded in 24-well plates at 2 × 105 cells/well. In all experiments negative control without LPG and positive control with cyclohexamide (50 μg/mL) were simultaneously conducted. The assays which needs cell lysates, the cell lysate was prepared by treating the cells with TritonX 100 (0.1%; 1 mL) and sonicating the contents for 20 s. The final suspension was centrifuged at 4000 rpm for 5 min for the removal of cell debris.
Poly herbal drug and preparation of poly-herbal extract
The traditional poly herbal anti cancer drug Le Pana Guliya (LPG) was obtained from the traditional medicinal practitioner Dr. Mahinda Ekanayake (Reg. number: 11797), No: 9, Moragahapitiya, Balagola, Kengalle, Kandy, Sri Lanka. Sample of 5 g of LPG from three different batches soaked in distilled water (100 mL) was kept in the rotary shaker for 48 h in an air tight dark bottle. The extract was then filtered through a layer of muslin cloth and filtrate was centrifuged at 3000 rpm for 15 min at 4 °C to remove any debris.
The supernatant was freeze dried, and stored at −20 °C in an air tight vial until used. Each different extracts were used for all the assays carried out in this study.
The freeze dried extract was reconstituted with distilled water for experimental purposes.
The drug extracts were prepared in triplicate and each experiment was performed in triplicates to each preparation. Cell viability was determined as percentage of the absorbance of the treated cells to that of un-treated cells.
Cell viability assay
The effect of aqueous extract of the LPG on the cell viability was determined by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The live cells reduce yellow MTT to purple formazan crystals by mitochondrial dehydrogenase enzyme . The cells were seeded in 24 well plates (NUNC, Denmark) and cultured over-night as mentioned above. The mono-layers of cells were treated with different concentrations of LPG extracts prepared in culture medium and incubated in a CO2 incubator at 37 °C for 24 h.
After 24 h, the growth medium was replaced with 1.0 mL of minimum essential media (MEM), and 100 µL of MTT (5 mg/mL in PBS). The cells were incubated at 37 °C for 4 h and the medium was carefully removed. The formazan product was dissolved in acidified isopropanol (0.05 M HCl in Isopropyl alcohol (IPA); 750 µL) and absorbance was read at 570 nm. Cell survival was expressed as a percentage of viable cells of treated samples to that of untreated cells (negative control).
Lactate dehydrogenase (LDH) activity
Cytotoxicity induced by the drug assessed by lactate dehydrogenase (LDH) leakage into the culture medium was carried out with slight modifications as described in Fotakis and Timbrell 2006 . Cells were seeded and treated as described in MTT assay. After 24 h incubation the culture medium was aspirated and centrifuged at 4000 rpm for 5 min and supernatant and the lysate were subjected to LDH assay using a commercially available, LDH assay kit (HUMAN).
Estimation of protein content
The protein content of the cell lysate was determined described by Lowry et al. 1951 , after treatment with LPG for 24 h. Briefly, sodium hydroxide (2 M, 100 μL) was added to the cell lysate (100 μL) and the mixture was incubated at 100 °C for 10 min. A mixture (1 mL) prepared by dilution (100:1:1) with Na2CO3 (2%), CuSO4·5H2O (1%) and sodium potassium tartrate (2%) was then added to the test solution and Folin–Ciocalteu reagent was added after 10 min. the samples were incubated for 30 min at room temperature in the dark. The absorbance was measured at 750 nm. Bovine serum albumin (BSA) was used for the calibration curve to determine the protein content of cell lysate. The percentage protein content of the treated cells to that of untreated cells was calculated using following equation.
HepG2, HeLa and CC1 cells at 70% confluence were treated with different concentrations of drug extracts for 24 h and observed under phase-contrast inverted fluorescence microscope (40×). The changes in morphology were compared with positive and negative controls.
Griess nitrite assay
The cell supernatant was used to assay nitric oxide production in cells, as explained by the method of Green et al. 1982 . Briefly 100 μL of the culture supernatant was incubated with 100 μL of Griess reagent (1% sulphanilamide in 0.1 mol/l HCl and 0.1% N-(1-naphthyl) ethylenediaminedihydrochloride at room temperature for 10 min.
The absorbance was measured at 540 nm. The nitrite content was calculated based on a standard curve constructed with NaNO2 and the nitrite content is expressed as nmoles.
Determination of cellular reduced glutathione (GSH) levels and effect of endogenous GSH on the cell viability LPG
The total reduced glutathione (GSH) content of the HepG2, HeLa and CC1 cells were determined using the methods described by in Padma et al. 2007  with slight modifications. The effect of exogenous GSH on the cell viability was also investigated in the presence of LPG. Briefly the cells were seeded as described earlier. The effect of endogenous GSH on cell viability was also determined after addition of GSH (25 μg mL) in the presence of LPG at same concentrations of EC50 obtained for MTT assays for respective cell lines. Negative control for each cell line was also carried out simultaneously. The cell viability was determined by MTT assay as described earlier. The GSH content was calculated based on a standard curve constructed with a series of reduced glutathione standards (0.5–3 µg/mL).
Measurement of mitochondrial membrane potential (MMP)
Rhodamine 123 was used to evaluate the changes in mitochondrial membrane potential as described previously . Briefly cells were incubated with LPG for 24 h. Cells were then washed with PBS (pH 7.4) and fixed with 70% ice cold ethanol.
Rhodamine 123 (20 μL; 10 μg/mL) was added to each well and incubated in the dark at 37 °C for 30 min. The cells were then washed gently with ice cold PBS twice and examined immediately using phase-contrast inverted fluorescence microscope (40×).
Caspase 3 activity
Caspase-3 activity of HepG2 and HeLa was assayed and compared with normal cells (CC1) according to the manufacturer’s instructions of Caspase-3/CPP 32 Colorimetric Assay Kit. Briefly the cells were seeded in a 12-well plate with a density of 2 × 106 cells/well, and treated with different concentrations of LPG in triplicates.
Ethidium bromide and acridine orange staining
Ethidium bromide and acridine orange staining was carried out to determine the induction of apoptosis by LPG according to the method described by Ribble et al. and Soysa et al. [13, 14] with slight modifications. Cells were seeded in 12 well plates and the confluent layer was treated with LPG at different concentrations for 24 h as described previously. The adherent cells were washed carefully with 1.0 mL of PBS followed by addition of 20 μL of the dye mix containing ethidium bromide (100 mg/mL) and acridine orange (100 mg/mL). Morphological changes were examined immediately using phase-contrast inverted fluorescence microscope (40×) under UV lamp. Live cells with normal nuclear chromatin exhibited green nuclear staining and the cells undergoing apoptosis showed orange to red . The changes in morphology were compared with positive and negative controls. Images were photographed using digital imaging system connected to microscope.
DNA fragmentation assay
The isolation of fragmented DNA was carried out according to the procedure of Kasibhatla et al.  with slight modifications.
Briefly, cells (2 × 106) were seeded in 12 well plates and treated with different concentrations (i.e. 0.5–2.5 for HepG2, 2.5–20.0 for HeLa and 50.0–500 μg/mL) of LPG for 24 h respectively. The cells were washed with PBS and trypsinized. The cell pellets were incubated with 20 μL lysis buffer (10 mM EDTA, 50 mMTris-HCl, 0.5% Sodium lauryl sarcosinate;pH 8) and 10 μL RNase A (final concentration 500 U/mL) at 37 °C for 4 h followed by digestion with proteinaseK for overnight at 50 °C. The samples were mixed with 8 μL of 6× DNA loading buffer. The DNA samples were subjected to electrophoresis on agarose gel (1.5%) in TBE buffer (89 mMTris-HCl, 89 mM Boric acid, 2 mM EDTA, pH 8.4) containing ethidium bromide (0.5 μg/mL The gel was run at 45 V and DNA was photographed using a UVI pro gel documentation system (UVItec UK.)
The results were expressed as mean ± standard deviation (Mean ± SD). The measurements were performed in triplicate and values shown are representative for at least three independent experiments. Least square linear regression analysis was applied using Microsoft excel to determine the EC50 values and for the calibration curves. R2 > 0.99 was considered as linear for the calibration curves. Significant differences of each test result were statistically analyzed using “Mann–Whitney U” test significances with 95% significance using SPSS version 16.
Results and discussion
MTT assay is a rapid colorimetric approach that widely used to determine cell growth and cell cytotoxicity. It measures mitochondrial activity through enzymatic reaction on the reduction of MTT to formazan .
EC50 values of MTT assay and LDH assay after 24 h incubation with LPG
EC50 (µg/mL) (n = 6)
2.72 ± 0.36*
19.03 ± 2.63**
213.07 ± 7.71
0.91 ± 0.03*
25.98 ± 0.59**
159.26 ± 3.09
After 24 h incubation with cyclohexamide (Positive control) at a concentration of 50 µg/mL, the tested cells showed percentage viability of 64.43 ± 3.01% for HepG2, 75.66 ± 1.06% for HeLa and 71.93 ± 2.66% for CC1 cells. In contrast, the percentage viability obtained for MTT assay at the same concentration of LPG (50 µg/mL) treatment was 17.2 ± 1.47%, 35.26 ± 2.9% and 79.01 ± 1.59% for HepG2, HeLa and CC1 cells respectively. The EC50 obtained for MTT indicate that the cytotoxicity of the crude extract against the cancer cell lines is within the limit of cytotoxicity (EC50 < 30 µg/mL), as reported by the American National Cancer Institute (NCI) over 72 h post exposure and it is beyond the limits for CC1 cells .
Leakage of cytoplasmic located enzyme LDH into the extracellular medium is measured in lactate dehydrogenase (LDH) assay. Previous studies suggest that LDH is a more reliable and accurate marker of cytotoxicity, since damaged cells is fragmented completely during the course of prolonged incubation with substances . Xia et al. reported that the intracellular LDH release to the medium is a measure of irreversible cell death due to cell membrane damage, where it is directly up regulate the subsequent induction of apoptosis .
It has been identified that the cellular stress conditions interfere with signaling pathways in protein synthesis . Protein content in the lysate was determined in all three cell types after 24 h exposure of LPG. The results showed that there was a decrease in total protein content inHepG2 and HeLa cells treated with LPG compared to untreated cells.
Previous reports showed that an excessive and unregulated NO synthesis has been implicated to regression of tumorgenicity and metastasis of tumor cells via alterations of the expression of apoptosis associated proteins . The elevated NO levels by LPG inhibit cell proliferation and trigger apoptosis . This is through DNA damage caused by DNA modification or DNA strand breakage ultimately leading to apoptosis .
Therefore, it is evident that generated nitrite from NO has played a significant role in inhibition of HepG2 and HeLa cell growth.
One of the most complex aspects in the regulation of cell death is the role of intracellular oxidation of biomolecules including proteins. It was initially proposed that cellular oxidative stress could be a general mediator of apoptosis . In fact, exposure to reactive oxygen and nitrogen species (RONS)such as hydrogen peroxide (H2O2) or nitric oxide (NO) induces cell death via apoptosis in different cell types . It was reported that the direct oxidant treatment and deregulated intracellular production of ROS are equally harmful to the cell and are countered by various antioxidant defenses. Among them, the tri-peptide GSH is the most rapid and abundant weapon against ROS and regulates the redox state of many other cellular constituents .
Effect of LPG on GSH levels in HepG2, HeLa and CC1 cells after 24 h treatment
Total GSH content (μg/mL)
10.15 ± 0.26
3.51 ± 0.23
5.72 ± 0.51
7.11 ± 0.11
2.22 ± 0.18
3.45 ± 0.32
9.41 ± 0.28
7.02 ± 0.75
6.06 ± 0.81
The results suggest that, depletion of GSH by LPG may contribute to the accumulation of RONS in the cells producing redox imbalance of the cells. This in turn leads to oxidation of biomolecules which are vital for cellular functions and membrane integrity causing cell death through stimulating of downstream events of apoptosis. Furthermore, reversing the cell death via neutralizing of ROS by exogenous GSH in the presence of LPG confirms that the induction of cell death is caused by oxidative stress.
The light microscopic photographs upon the treatment with LPG indicate prominent features of apoptosis. Similarly in the presence of high levels of NO and depletion in cellular GSH suggested that the LPG may exert its cytotoxicity via induction of apoptotic pathway.
Changes in the ∆ψm have been originally hypothesized to be early, coerce events in the apoptotic signaling pathway . It was reported that the mitochondrial permeability transition pore (PTP) which act as the “mega-channel” that results in the release of certain mitochondrial apoptogenic factors in some cell types during apoptosis . LPG results in opening up of PTP leading to activation of caspase-3 through cascade of intracellular events resulting cell membrane blebbing, nuclear condensation and DNA fragmentation as a results of depletion of ∆ψm.
Induction of apoptosis in HepG2 and HeLa cells by LPG were observed in the presence of AO/EB staining. Acridine orange (AO) permeates both live and dead cells and stains DNA and makes the nucleus appear green while ethidium bromide (EB) is only taken up by cells with damaged cell membranes . Thus, live cells will be uniformly stained green, apoptotic cells will be stained as orange or displayed orange fragments, nuclear fragmentation, presence of apoptotic bodies and blebbing when observed under fluorescence microscope depending on the degree of loss of membrane integrity.
This was further confirmed by the DNA fragmentation assay indicating a unique ladder banding pattern. With the activation of Caspase-3 it cleaves inhibitor of caspase activated DNase (ICAD), promoting release of active caspase-activated DNase (CAD) , The activated CAD then cleaves oligonucleosomal DNA at the inter-nucleosomal linker sites yielding DNA fragments in multiples of 180 base pairs .
Based on the results we can conclude that LPG stimulates tumor specific oxidative stress. Activation of caspases through mitochondrial impairment caused by oxidative stress stimulates downstream events of apoptosis leading to DNA fragmentation and cell death in HepG2 and HeLa cells. LPG is more effective in inducing apoptosis in HepG2 cells and minimal cytotoxicity towards normal cell line CC1. The potent anticancer and apoptotic effects of LPG observed in the present study provide the scientific proof of the traditional knowledge in using LPG as an anticancer agent.
NDAW performed all of the experiments and analyzed the data. PS designed, and supervised the study. AAPK and CMI supervised part of the study. ME produce and supplied the poly herbal drug. All authors read and approved the final manuscript.
The financial support by World Class University Research Grant No. AP/3/2012/CG/10 awarded to Professor Preethi Soysa is acknowledged.
The authors declare that they have no competing interests.
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