Apoptosis Induction and S-Phase Cell Cycle Blockade in Human Lung Adenocarcinoma Cell Line (A549) by Chlorophytum comosum (Thunb.) Jaques

Apoptosis Induction and S-Phase Cell Cycle Blockade in Human Lung Adenocarcinoma Cell Line (A549) by Chlorophytum comosum (Thunb.) 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Jaques Shehla Adhami, Humaira Farooqi, Asrar Ahmad Malik This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5897484/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Methods The ethanolic roots (CCRE) and leaves (CCLE) extracts were interrogated for their apoptotic potential against human lung adenocarcinoma cell line (A549) using DNA fragmentation, Annexin V-FITC/PI staining apoptosis assay and cell cycle analysis using flow cytometry. Results Our results revealed significant DNA damage and apoptosis induced cell death in A549 cell line on treatment with active concentrations (40 µg/ml and 80 µg/ml) of the ethanolic extracts with S phase cell cycle arrest. Conclusions This is the first study demonstrating the apoptosis inducing potential of chemically characterized bioactive compounds present in ethanolic leaves and roots extracts from Chlorophytum comosum against non-small cell human lung adenocarcinoma cell line. The study concludes that Chlorophytum comosum can be a potential candidate for the natural bioactive compounds that can be isolated, characterized and clinically evaluated for the development of novel naturally derived anti-cancer drugs against lung cancer. Natural Product Chemistry Cell Cycle & Proliferation Cell Survival and Cell Death Apoptosis A549 Chlorophytum comosum Lung Cancer Spider Plant Phytoterapeutics Figures Figure 1 Figure 2 Figure 3 1. Introduction Lung cancer is the second most aggressive type of cancer contributing highest (21%) of all cancer deaths [ 1 ]. Unlike other cancer types, where often the risk of cancer occurrence is influenced by genetic and epigenetic factors; lung cancer in majority, is a detrimental consequence of smoking habits observed among the human population and continued direct exposure of the harmful carcinogens present in the environment. Lung cancer incidence and mortality rates are tightly linked to cigarette smoking patterns [ 2 ]. Another challenge associated with lung cancer is its delayed detection mostly at advanced stage. Approximately 44% of people have advanced stage disease at diagnosis [ 2 ]. According to American Cancer Society, in US alone, approximately 101,300 (81%) of the 125,070 lung cancer deaths in 2024 will be caused by direct cigarette smoking with an additional mortality of 3500 which will be caused by second hand smoke [ 1 ]. Despite the availability of modern chemotherapeutic interventions like personalized immunotherapy, chemotherapy, radiotherapy and surgical methods, the 5-year relative survival rate for all stages combined in lung cancer is 25% [ 1 ]. The side effects of lung cancer treatment by these approaches render patients to look for alternative therapies that may help in improving the quality of life and naturally delaying the recurrent episodes of severe side effects as the disease progresses. Several forms of complementary and alternative medicine practiced under different traditional medicinal systems are now under investigation for its potential role in curbing and combating cancer [ 3 , 4 ]. In China, Traditional Chinese Medicine (TCM) has been widely accepted as a mainstream form of complementary and alternative therapy for cancer patients [ 5 , 6 , 7 ]. A large number of studies on Traditional Chinese Medicine are elucidating the role of the herbs at the molecular level to understand its impact on cancer signaling pathways [ 8 , 9 , 10 ]. Chlorophytum comosum (Thunb.) Jaques belongs to family Lilliaceae and genus Chlorophytum [ 11 ]. It is commonly known as Spider plant and is worldwide popular for its ornamental value. In Traditional Chinese Medicine, it is used for the treatment of fractures, burns and respiratory ailments such as bronchitis and asthma [ 12 , 13 ]. In India, roots of Chlorophytum comosum are used as a substitute for another medicinal Chlorophytum species ( Chlorophytum borivilianum ) for preparation of an important class of ayurvedic drugs known as Rasayna [ 14 ]. Besides, several other species belonging to genus Chlorophytum are well known for their pharmacological properties [ 15 , 16 ]. The genus Chlorophytum is predominantly rich in saponins and has been extensively studied for its cytotoxic properties [ 17 ]. However, as far as Chlorophytum comosum is concerned not much work has been done to discover its potential as an anticancer herb. Preliminary findings from earlier studies indicates its cytotoxic potential against selected cancer cell lines, however, these studies were only limited to initial cytotoxicity screening without deciphering the mechanism of action [ 18 , 19 ]. Saponins isolated from roots of Chlorophytum comosum have been found to exhibit cytotoxic and anti-tumour promoter activity in HeLa cancer cell line [ 18 ]. In an another important study, DNA damage in four different human cancer cell lines viz. HeLa, CCRF-HSB-2, HL-60 and U937 cells under the effects of n-butanol root fraction from Chlorophytum comosum was demonstrated using qualitative DNA fragmentation assay and the Terminal Deoxynucleotidyl Transferase (TdT) mediated biotin dUTP Nick End Labeling (TUNEL) method [ 19 ]. In a study, methanolic leaf extract of Chlorophytum comosum was evaluated for its chemical profile and its different sub fractions were explored for the bioactivity. The study revealed steroids and isoprenoid as the most abundant compounds present in methanolic leaf extract with higher percentage of unsaturated fatty acids and neophytadiene as the second highest abundant compound. Biological assessment of its sub fractions viz. chloroform, n-hexane, n-butanol and water revealed significant in vitro antioxidant and selective cytotoxic activity against HeLa cancer cell lines [ 20 ]. Recently, progressive neuroprotective efficacy of roots and leaves extracts of Chlorophytum comosum at the concentrations of 60 µg/ml and 90 µg/ml were demonstrated against glutamate excitotoxicity in a culture of rat cerebellar neurons retrieved from 7–9 day old rats [ 21 ]. Chlorophytum comosum inspite of having a strong Ethnopharmacological base and as an important ingredient in nutritional/health supplement in different traditional medicinal systems such as Traditional Chinese Medicine (TCM), African Traditional Medicine (ATM) and Ayurveda is yet unexplored on scientific grounds for its therapeutic potential [ 12 , 13 , 22 , 23 , 14 ]. Moreover, with view of its traditional usage for treatment of respiratory ailments, so far, no biological activity has been reported to explore its antiproliferative efficacy against lung cancer. Thus, keeping in view of the earlier literature and Ethanomedical lead, the present study aimed to further investigate the anticancer potential of ethanolic roots and leaves extract of Chlorophytum comosum against human lung adenocarcinoma cell line (A549) by understanding the mode of cancer cell death prompted by the extracts. 2. Materials and Methods 2.1 Chemicals and Reagents Solvents used in the study i.e. n-Butanol, Petroleum Ether, Water, were of HPLC/LC-MS grade and were purchased from Merck (Darmstadt, Germany). Ethanol (China grade) and Hydrogen peroxide solution was purchased from local commercial supply (Thomas Baker, India). Cell culture media DMEM with phenol red (#1932403), RPMI-1640 (# 1898961), Fetal Bovine Serum (#10438034), Trypsin–EDTA solution with phenol red (#1897336) and antibiotic solution PenStrep (#192493) were purchased from Gibco USA. MTT reagent (#MICB8173V), Propidium iodide (# P4864), RNase A, Standard Doxorubicin and Colchicine were obtained from Sigma Aldrich USA. FITC-Annexin V apoptosis detection kit was obtained from Invitrogen USA (# V-13242). Standard Vinblastine sulphate was purchased from Cipla (India). Sheath buffer was purchased from BD Biosciences-IN. All the cell lines used in the study were procured from ATCC (USA). 2.2 Plant Material Collection Mature roots and leaves part of Chlorophytum comosum ; cultivated for a period of 180 days were harvested in June 2016 from Herbal Garden, Jamia Hamdard, New Delhi. The plant parts collected were healthy and disease free. The plant samples were identified by the botanist Dr. Sunita Garg (NISCAIR, New Delhi) and the voucher specimens bearing no. NISCAIR/RHMD/CONSULT/ 2016/2975-02 were deposited in the herbarium. 2.3 Drug Preparation and Treatment for MTT Assay For MTT assay, the semi dried extracts were weighed and initially prepared in DMSO with a final concentration of DMSO < 0.1%. The samples were two fold serially diluted to 6 different concentrations (10–320 µg/ml) [Stock conc. 3.2 mg/ml; Working conc. 0.32 mg/ml] using incomplete DMEM medium. Vinblastine sulphate served as positive control with 6 different test concentrations (3.12–100 µM) [Stock conc.1.2 mM] [ 24 ]. Cells with 1% DMSO served as vehicle control, while, cells with only media served as negative control. Time duration for treatment was 24 hrs. 2.4 Cell Lines Maintenance Human lung adenocarcinoma cell line (A549) [CCL-185] and human normal lung cell line (L-132) [CCL-5] were procured from the American Type Culture Collection (Manassas, VA, USA). The cell lines obtained were pre- authenticated (molecularly characterized). Cell lines quality control specifications and authentication documentation were confirmed from the available information on the ATCC website as reference. Cell lines were maintained in DMEM/RPMI-1640 medium supplemented with 10% heat inactivated FBS and 1% penicillin/streptomycin solution with 5% CO 2 supply at 37° C. 2.5 Experimental 2.5.1 Preparation of Plant Extracts Ethanolic extracts of roots (CCRE) and leaves (CCLE) of Chlorophytum comosum were prepared using hot continuous Soxhlet extraction method. Briefly, fresh plant materials were collected and washed to remove debris, shade dried under sunlight for seven consecutive days to remove the moisture content until no change in weight was observed. The grinded plant material (10 gm) each was defatted using petroleum ether (1:15 w/v), dried and suspended in ethanol (150 mL), and extracted using Soxhlet apparatus for 24 hrs at 50 0 C. The extracts were then collected, filtered, and partitioned using n-butanol until the upper layer became colorless. The upper layer was further collected, pooled and concentrated on a rotary evaporator at 55 0 C and 55 mbar pressure until the semi-dried substance was obtained. The semi-dried substance was further lyophilized to remove the traces of the solvent. The samples were then stored in airtight vials at 4 0 C till further use. 2.5.2 Cell Cytotoxicity Assay by MTT Representing methodology from our previous data for continuation of the study [ 25 ], the cytotoxic activity in terms of % cell inhibition by roots ethanolic (CCRE) and leaves ethanolic (CCLE) extracts were measured using cell based colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction MTT assay with the treatment time of 24 hrs [ 26 ]. Briefly, monolayer cell culture (A549 & L-132) grown in T 25 cell culture flask was trypsanized using trypsin EDTA solution with phenol red and collected as pellet after centrifugation at 1000 rpm in 1 ml of complete medium. The total number of viable cells per ml was then counted with the help of Trypan blue dye (1:1 ratio with dilution factor 2) using hemocytometer. The cell count was adjusted to 40,000 cells/well, and to each well of the 96 well microtiter plate, 100 µl of the diluted cell suspension containing 40,000 cells was added. After 24 hrs, media was removed, cells were washed once with fresh medium and further treated with 100 µl of varying concentrations of extracts (10–320 µg/ml) and Vinblastine (3.12–100 µM) for 24 hrs. Subsequently, post treatment, 10 µL of MTT dye (0.5 mg/mL) was loaded inside each well and incubated further for 4 hrs at 37 0 C in an incubator supplied with 5% CO 2 . MTT solution was removed and crystals formed were then dissolved in DMSO. Finally, absorbance was taken at 570 nm using microplate reader (Spectra Max, USA). The % cell inhibition was measured by using the following formula (Eq A.1). The concentration at which the test drug inhibited cell growth by 50% i.e. inhibitory concentration (IC 50 ) was generated from the dose-response curves by nonlinear regression analysis using Graph Pad Prism software (8.1). % Cell Inhibition by CCLE /CCRE extracts = [(C – S) X 100] / C ………. (Eq A.1) C = Absorbance of control; S = Absorbance of samples. 2.5.3 Determination of Test Doses Based on IC 50 Values The half maximal inhibitory concentration (IC 50 ) is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process like cell growth by half. To determine the apoptosis inducing effects of Chlorophytum comosum leaves ethanolic extract (CCLE) and roots ethanolic extract (CCRE), the active lower and higher doses were set corresponding to the determined IC 50 values of each extract from our previously published data [ 25 ]. The doses ranges selected for CCRE with an IC 50 value of 68.68 µg/ml and CCLE with an IC 50 value of 61.19 µg/ml were 40 µg/ml & 80 µg/ml respectively. Both the IC 50 values were falling between these two upper and lower doses selected for the study. 2.5.4 DNA Fragmentation Assay Qualitative estimation of apoptosis inducing effects of the Chlorophytum comosum ethanolic extracts were measured using DNA fragmentation ladder assay. The formation of distinct DNA fragments of oligonucleosomal size (180–200 bp length) is a biochemical hallmark of apoptosis in many cells [ 27 ]. These DNA fragments can be extracted from the cells post treatment and visualized using agarose gel electrophoresis method. Briefly, A549 cells seeded in DMEM medium at a concentration of 8 x 10 5 cells per 35 mm dish, incubated at 37 o C, 5% CO 2 were treated with two concentrations i.e. (40 µg/ml and 80 µg/ml ) of each sample (CCRE & CCLE) for 24 hrs. Hydrogen peroxide served as control [ 28 ]. Total genomic DNA was taken out and DNA damage was evaluated using 1% agarose gel [ 29 ]. DNA ladder of 1500 bp served as marker ladder to determine the apoptotic fragments. 2.5.5 Cell Cycle Analysis by Propidium Iodide Cell cycle analysis was performed by PI (Propidium iodide) staining method using flow cytometry [ 30 , 31 ]. Briefly, A549 cells (8 x 10 5 cells/ml) cultured using 2 ml of DMEM medium in 6 well plates were treated with desired concentrations (40 µg/ml and 80 µg/ml) of test extracts (CCLE & CCRE) for 24 hrs. Post incubation, the medium was discarded and cells were gently scrapped using BD cell scrapper, transferred to RIA vials and centrifuged at 4500 rpm for 5 min at 4 0 C. The supernatant was discarded carefully retaining the cell pellets. Cell pellets were further washed twice using 1X PBS and fixed by resuspending in 300 µl of Sheath fluid followed by addition of 1mL of chilled 70% ethyl alcohol drop by drop with continuous gentle shaking and another 1 mL of chilled 70% ethyl alcohol added at once. The samples were stored at 4 0 C. Subsequently, cell pellets were washed twice with 2 ml of ice cold 1X PBS and then suspended in 450 µl of sheath fluid containing 16 µl PI [Stock conc. 0.05 mg/ml] and 16 µl RNaseA [Stock conc. 2 mg/ml; Working conc. 1 mg/ml] and incubated for 30 min in dark. The percent DNA content in various cycle phases in treated versus negative (untreated) control cells were determined using FACS Caliber instrument (BD Biosciences) with Cell Quest software (BD Biosciences, San Jose, CA, USA). Colchicine treated A549 cells (25 µM) served as positive control [ 32 ]. 2.5.6 Apoptosis Detection Using PI/Annexin V-FITC Staining Apoptosis detection was performed using a commercial kit (Invitrogen V-3242) following the manufacturer's recommendations. Briefly, 8 x 10 5 A549 cells/ml plated in DMEM medium were treated with 40 and 80 µg/ml of CCLE and CCRE extracts for 24 hrs. The cells were then harvested, centrifuged at 4500 rpm for 5 min at 4 0 C and washed twice with ice-cold 1X PBS. The pellets were suspended with the 1X binding buffer (100 µL) and then PI (10 µL) [Stock conc. 1mg/ml] and Annexin V-FITC (3 µL) was added. The suspension was incubated in dark for 10 min at room temperature (RT). Before flow cytometry analysis, 100µL of 1X binding buffer was further added to the suspension. Data from 10,000 cells per sample were collected and analyzed by flow cytometry using a FACS Caliber instrument with Cell Quest software (BD Biosciences, San Jose, CA, USA). The results were compared with the untreated negative control A549 cells. Doxorubicin treated A549 cells (25 µM) served as positive control [ 33 ]. 2.5.7 Statistical Analysis The statistical analysis was performed with one way analysis of variance (ANOVA) to calculate the statistical significance with p < 0.05 as considered significant. The inhibitory concentration (IC 50 ) values were calculated by nonlinear regression curve (log inhibitor vs. normalized response variable slope) with the use of Graph pad Prism version 8.1 for Windows (Graph pad Prism Software, San Diego, CA, USA). All results were expressed as mean ± standard deviation. 3. Results 3.1 Selective Cytotoxicity of Chlorophytum comosum against Human Lung Adenocarcinoma (A549) Cell Line by MTT Assay Based on our earlier published data [ 25 ] on antiproliferative effects of Chlorophytum comosum , dose dependent selective cytotoxic activity towards A549 lung cancer cell line with an IC 50 values of 68.68 µg/mL and 61.19 µg/mL were demonstrated by both roots ethanolic (CCRE) and leaves ethanolic (CCLE) extracts respectively (Table 1) . Leaves extract showed better cytotoxic response as compared to roots extract with lesser IC 50 values. In positive control taken as Vinblastine sulphate, maximum inhibitory effect with an IC 50 value of 18.79 µM and 20.71 µM was observed in A549 and L-132 respectively [ 25 ]. While in case of normal human lung cell line (L-132), the IC 50 values were not calculated due to lesser percentage inhibition i.e.< 50%, indicating non-significant activity. For the present study, the test doses were selected as two active concentrations i.e. 40 µg/ml and 80 µg/ml for both roots ethanolic and leaves ethanolic extracts representing the lower and upper ranges between the determined IC 50 values to carry out the further investigations. 3.2 Chlorophytum comosum Leaves Ethanolic (CCLE) and Roots Ethanolic (CCRE) Extracts Promoted Nuclear DNA Fragmentation in A549 Cell Line One major characteristic feature of apoptotic mode of cell death is the extensive morphological and biochemical changes that ensure the vanishment of dying cells without affecting and harming the other neighboring cells. In contrast, during necrosis mode of cell death, the cell gets destroyed leaving the traces of cellular contents that activate the chain reaction of inflammation in the cellular microenvironment. Formation of short distinct fragments of DNA of 180–200 bp length visualized on agarose gel represents DNA damage and is considered as a biochemical hallmark of apoptosis process [ 27 ]. The apoptosis induction in A549 human lung adenocarcinoma cell line by the roots ethanolic and leaves ethanolic extracts of Chlorophytum comosum was qualitatively reported using gel based DNA fragmentation assay. A dose dependent internucleosomal fragmentation pattern was observed in A549 cells on 1.8% agarose gel stained with ethidium bromide when treated with CCLE extract at the concentrations of 40 µg/ml and 80 µg/ml for 24 hrs. Fine smearing throughout the gel represented significant DNA damage. Similarly in case of CCRE extract, significant DNA damage was observed at both doses, however, compared to leaves (CCLE) extract, the damage in roots extract was moderately recorded at 80 µg/ml. Hydrogen peroxide taken as positive control was used to compare the fragmentation pattern. Overall, these results suggested that both extracts induced DNA fragmentation in A549 cell line indicating DNA damage by apoptosis Fig. 1 (a & b). 3.3 Chlorophytum comosum Induced Early and Late Apoptotic Cell Death in A549 Cells The apoptotic effect of Chlorophytum comosum roots ethanolic (CCRE) and leaves ethanolic (CCLE) extracts was confirmed using Annexin- FITC/PI staining by evaluating the stages of cell death being early apoptotic, late apoptotic and necrotic. Apoptosis initiation is markedly represented by the acquisition of surface changes by dying cells such as the expression of thrombospondin binding sites, loss of sialic acid residues and exposure of phospholipids like phosphatidylserine (PS) [ 34 ]. Early apoptotic events start with the translocation and exposure of phosphatidylserine to the outer layer of the membrane with intact cell membrane. True apoptotic cells can be quantitated using Annexin V dye which preferentially binds to negatively charged phospholipids like PS in the presence of Ca 2+ ; the events of which are recorded in the form of fluorescence signal by flow cytometry. Staining cells simultaneously with FITC-Annexin V and the Propidium iodide allows the discrimination of intact cells by early apoptotic, late apoptotic or necrotic cells [ 34 ]. Apoptosis induction studies using Annexin V-FITC/PI staining demonstrated the occurrence of apoptosis in A549 cells after treatment with CCLE and CCRE extracts for 24 hrs. Dose dependent decrease in cell viability with varying % of cells in early apoptotic, late apoptotic and necrosis stages were recorded Fig. 2a. The 40 and 80 µg/ml treatment of leaves extract (CCLE) induced early apoptosis by 5.69%, 1.95%, and late apoptosis by 10.19%, 7.21% respectively. The roots extract (CCRE) treatment at 40 µg/ml and 80 µg/ml has induced early apoptosis by 9.67%, 5.38% and late apoptosis by 3.98%, 8.15% respectively. Necrotic cells were found to be 13.71%, 30.32% and 4.77%, 16.93% at 40 µg/ml and 80 µg/ml in CCLE and CCRE extracts respectively Fig. 2b. In control untreated A549 cells, 91% of viable cell remained intact with 8.32% early apoptotic cells with negligible late apoptotic cells i.e. 0.21% and no necrotic detected cells Fig. 2b. These results indicate that the death of A549 cells induced by Chlorophytum comosum was mediated by apoptosis. Doxorubicin used as positive control (standard), detected 14.44% early apoptotic, 30.66% late apoptotic, and 12.21% necrotic cells with 42.69% of viable cells Fig. 2b. 3.4 Chlorophytum comosum Arrested Cell Cycle Progression at S Phase The effects of leaves ethanolic (CCLE) and roots ethanolic (CCRE) extracts of Chlorophytum comosum were seen on cell cycle progression analyzed using PI staining detected by flow cytometry method. The treatment of A549 cells with CCLE and CCRE extracts for 24 hr at two different dosages (40 µg/ml and 80 µg/ml) led to the cell cycle arrest at S phase Fig. 3a. The cell cycle arrest was found to be in dose dependent manner with two fold change which was observed in both the cases from lower to higher concentrations, however, the leaves extract was found to be most effective in accumulating and arresting cells at S phase followed by the roots extract. CCLE extract has shown dose dependent S phase arrest of 23.23% and 42.86% at 40 µg/ml and 80 µg/ml respectively, compared to control untreated cells which showed only 5.90% arrest in A549 cells at S phase. While in CCRE extract, S phase arrest was 19.64% at 40 µg/ml and 38.03% at 80 µg/ml. Standard Colchicine at 25 µM showed 51.33% S phase arrest and 5.08% G2 arrest in A549 cells Fig. 3b. 4. Discussion The exploration of anticancer drugs from plant source is rapidly evolving with various high ends in vitro and in silico screening techniques which helps in the preliminary identification of the active target biomolecules that may serve as lead candidates in anticancer therapy [ 35 , 36 ]. Several phytochemicals from plants have been found to possess anticancer activity by virtue of their ability in modulating the cell growth regulatory mechanisms i.e. cell cycle regulation and by inducing apoptosis in cancer cells when treated [ 37 , 38 ]. The apoptosis inducing ability of the phytoconstituents in immortal cancer cells is thus, a remarkable attempt to control the progression and spread of the cancer. This is achieved by the manipulation and activation of various cancer signaling pathways at the molecular level [ 36 , 39 ]. In the present study, an attempt has been made to evaluate the anticancer potential of ethanolic roots and leaves extracts from Chlorophytum comosum against A549 human non-small cell lung carcinoma cell line by determining the mode of cell death and its underlying mechanism. Chlorophytum comosum is reported to be used in treatment of various respiratory ailments in Traditional Chinese Medicine (TCM) and has been found to be useful in relief from bronchitis and asthma [ 12 , 13 ]. Saponins are the chief active constituents found in Chlorophytum comosum and possess cytotoxic potential against cancer cell lines [ 17 – 19 ]. However, till date no scientific studies were carried out with regard to respiratory ailments that could also justify its ethnomedical importance. To our best of knowledge, our study for the first time evaluated the apoptosis inducing potential of Chlorophytum comosum against lung cancer using in vitro human lung adenocarcinoma cell line (A549) model which is an extensively studied model for human lung epithelial cancer and possesses the high metastatic potential [ 40 ]. This work is in continuation of our previously published work, where cytotoxicity studies by MTT assay revealed significant antiproliferative effects by chemically characterized ethanolic roots and leaves extracts selectively towards A549 cell line. While, no antiproliferative effects of the extracts at the same tested concentrations were observed against normal human lung cell line L-132. On the other hand positive control Vinblastine exhibited significant cytotoxicity towards both A549 and L-132 cell lines. In the present study, based on the earlier demonstrated IC 50 values, two active concentrations of the extracts were selected and we further investigated the markers of cell death process to ascertain the bioactivity of Chlorophytum comosum. We observed a systematic mode of cell death via apoptosis in A549 cells under the effect of the treatments. The early markers in apoptosis induced cell death involve internucleosomal DNA damage, cell membrane disruption and expression of Phosphatidylserine on outer membrane surface [ 34 ]. In our study, all these changes were systematically evident in A549 lung cancer cells post treatment. Annexin V-FITC/PI flow cytometry studies revealed increase in early apoptosis, late apoptosis and necrotic cells after CCLE and CCRE extracts treatment for 24 hrs with dose dependent decrease in the cell viability. DNA fragmentation studies indicated the DNA damage initiated by the treatment in response to non-treated control cells where no such activity was observed. However, the potency of each extract exhibited varied response at different concentrations selected for the study. CCLE extract exhibited greater potential as compared to CCRE extract. Another aspect associated with cancer control and progression is cell stage specific growth during which many proteins are altered and leads to malignant conditions. Studies reveals that plant derived drugs block cell cycle progression at various stages of cell cycle such as G0/G1, S or G2/M phases to induce apoptotic cell death [ 40 , 41 , 42 ]. Our study reveals the arrest of cell cycle of malignant A549 cells when treated with CCLE and CCRE extracts at S phase Fig. 3 . The growth arrest and apoptosis inducing features of the plant extracts have been found significant in number of studies. In a study conducted by Yerlikaya et al. [ 43 ], ethyl acetate extract from Lotus corniculatus exhibited significant apoptotic driven cell death via increased intranuclear influx of Ca2 + in MDA-MBA-231 breast cancer cell line. In a similar findings by Lee et al. [ 44 ], ethanolic extract from Calotropis gigantean induced apoptosis through the activation of extrinsic and intrinsic pathways, cell cycle arrest, and ROS generation in A549 and NCI-H1299 lung cancer cell lines. In a study conducted by Samarghandian et al. [ 45 ], antineoplastic potential of a plant derived Thymoquinone was assessed using flow cytometry studies on A549 cell line and were found to significantly up regulate the apoptotic modulators viz. p53 , caspase 3 and 9 with significantly elevated Bax/Bcl-2 ratio thereby, concluding initiation of apoptosis in A549 cells via caspase cascade dependent pathways. Thus, with insights from earlier literature and following the lead of our previous work where significant cytotoxic effects of ethanolic extracts of Chlorophytum comosum were evident on human lung carcinoma cell lines; the present study confirms the antiproliferative and antitumor effects of CCLE and CCRE extracts in A549 lung cancer cell line. This the first report of lung cancer cell toxicity and programmed cell death via apoptosis pathway in A549 cell line by Chlorophytum comosum ethanolic roots and leaves extracts. Such data indicates that Chlorophytum comosum have therapeutic phytochemical leads as apoptosis inducers in lung cancer cells that can be further isolated, characterized and studied and their potency as anticancer agents could be explored in the management of lung cancer. However, persuasive investigation is required to further characterize and elucidate the cancer signaling pathways involved in the bioactivity of active constituents from Chlorophytum comosum . 5. Conclusions Active constituents from roots and leaves parts of Chlorophytum comosum possess significant cytotoxic and apoptosis inducing potential against A549 lung cancer cell line suggestive of its anticancer potential. However, further in line research, based on in vivo and clinical studies will reveal novel bioactive leads from Chlorophytum comosum (Thunb.) Jaques. Abbreviations CCRE Chlorophytum comosum roots ethanolic extract CCLE Chlorophytum comosum leaves ethanolic extract FBS Fetal Bovine Serum PBS Phosphate Buffer Saline A549 Human Lung Adenocarcinoma Cell Line Declarations Financial Support This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Data Availability Statement The raw data that support the findings of this study are available from the corresponding author [Dr. Humaira Farooqi] upon reasonable request. Acknowledgements The authors are thankful to Professor M.Z. Abdin (Department of Biotechnology, Jamia Hamdard) for providing facilities for sample preparations. Mr. Hashmat (Herbal Garden, Jamia Hamdard) for supervising and providing plant samples. Dr. Sunita Garg and Professor M.P. Sharma for authentication of plant samples and Hamdard National Foundation for granting research fellowship to Dr. Adhami. The kind guidance and technical assistance of Skanda Life Sciences, Bangalore is highly appreciated. Declaration of Interest The authors report there are no competing interests to declare. References Siegel RL, Giaquinto AN, Jemal A (2024) Cancer Statistics, 2024. CA Cancer J Clin 74:21–29. https://doi.org/10.3322/caac.21820 Siegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer Statistics, 2023. CA Cancer J Clin 73(1):17–48. https://doi.org/10.3322/caac.21763 Knecht K, Kinder D, Stockert A (2020) Biologically Based Complementary and Alternative Medicine (CAM) Use in Cancer Patients: The Good, the Bad, the Misunderstood. 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Anticancer Agents Med Chem 21(13):1697–1707. https://doi.org/10.2174/1871520620666201123085300 Barltrop JA, Owen TC, Cory AH, Cory JG (1991) 5-(3-Carboxymethoxyphenyl)-2-(4,5-Dimethylthiazolyl)-3-(4-Sulfophenyl) Tetrazolium, Inner Salt (MTS) and Related Analogs of 3-(4,5-Dimethylthiazolyl)-2,5-Diphenyltetrazolium Bromide (MTT) Reducing to Purple Water-Soluble Formazans as Cell-Viability Indica. Bioorg Med Chem Lett 1:611–614. https://doi.org/10.1016/S0960-894X(01)81162-8 Bortner CD, Oldenburg NBE, Cidlowski JA (1995) The Role of DNA Fragmentation in Apoptosis. Trends Cell Biol 5(1):21–26. https://doi.org/10.1016/S0962-8924(00)88932-1 Takada M, Noguchi A, Sayama Y, Kurohane KY, Ishikawa T (2011) Inositol 1,4,5-Trisphosphate Receptor-Mediated Initial Ca 2+ Mobilization Constitutes a Triggering Signal for Hydrogen Peroxide-Induced Apoptosis in INS-1 β-Cells. Biol Pharm Bull 34(7):954–958. https://doi.org/10.1248/bpb.34.954 Li Z, Yu J, Liu L, Wei Z, Ehrlich ES, Liu G, Li J, Liu X, Wang H, Yu XF, Zhang W (2014) Coxsackievirus A16 Infection Induces Neural Cell and Non-neural Cell Apoptosis In Vitro . PLoS ONE 9(10):e111174. https://doi.org/10.1371/journal.pone.0111174 Darzynkiewicz Z, Bedner E, Smolewski P (2001) Flow Cytometry in Analysis of Cell Cycle and Apoptosis. Semin Hematol 38(2):179–193. https://doi.org/10.1016/S0037-1963(01)90051-4 Darzynkiewicz Z, Huang X, Zhao H (2017) Analysis of Cellular DNA Content by Flow Cytometry. Curr Protoc Cytom 82. 7.5.1–7.5.20 Thomas E, Gopalakrishnan V, Hegde M, Kumar S, Karki SS, Raghavan SC, Choudhary B (2016) A Novel Resveratrol Based Tubulin Inhibitor Induces Mitotic Arrest and Activates Apoptosis in Cancer Cells. Sci Rep 6:34653. https://doi.org/10.1038/srep34653 Ho YF, Karsani SA, Yong WK, Abd Malek SN (2013) Induction of Apoptosis and Cell Cycle Blockade by Helichrysetin in A549 Human Lung Adenocarcinoma Cells. Evidence-Based Complement Altern Med 3:857527. https://doi.org/10.1155/2013/857257 Morrone S (1998) Annexin V. Cytometry CD Rom Series V.4. Purdue Cytometry, USA Boyd MR (1997) The NCI In Vitro Anticancer Drug Discovery Screen. In: Teicher BA (ed) Anticancer Drug Development Guide, Cancer Drug Discovery and Development. Humana, Totowa, NJ, pp 23–42. https://doi.org/10.1007/978-1-4615-8152-9_2 . Chunarkar-Patil P, Kaleem M, Mishra R, Ray S, Ahmad A, Verma D, Bhayye S, Dubey R, Singh HN, Kumar S (2024) Anticancer Drug Discovery Based on Natural Products: From Computational Approaches to Clinical Studies. Biomedicines 12(1):201. https://doi.org/10.3390/biomedicines12010201 Iqbal J, Abbasi BA, Mahmood T, Kanwal S, Ali B, Shah SA, Khalil AT (2017) Plant-Derived Anticancer Agents: A Green Anticancer Approach. Asian Pac J Trop Biomed 7(12):1129–1150. https://doi.org/10.1016/j.apjtb.2017.10.016 Rayan A, Raiyn J, Falah M (2017) Nature is the Best Source of Anticancer Drugs: Indexing Natural Products for their Anticancer Bioactivity. PLoS ONE 12(11):e0187925. https://doi.org/10.1371/journal.pone.0187925 Peng F, Liao M, Qin R, Zhu S, Peng C, Fu L, Chen Y, Han B (2022) Regulated Cell Death (RCD) in Cancer: Key Pathways and Targeted Therapies. Sig Transduct Target Ther 7:286. https://doi.org/10.1038/s41392-022-01110-y Garcia-de-Alba C (2021) Repurposing A549 Adenocarcinoma Cells: New Options for Drug Discovery. Am J Respir Cell Mol Biol 64(4):405–406. https://doi.org/10.1165/rcmb.2021-0048ED Bai J, Li Y, Zhang G (2017) Cell Cycle Regulation and Anticancer Drug Discovery. Cancer Biol Med 14(4):348–362. https://doi.org/10.20892/j.issn.2095-3941.2017.0033 Banerjee S, Nau S, Hochwald SN, Xie H, Zhang J (2023) Anticancer Properties and Mechanisms of Botanical Derivatives. Phytomedicine Plus 3(1):100396. https://doi.org/10.1016/j.phyplu.2022.100396 Yerlikaya S, Baloglu MC, Diuzheva A, Jeko J, Cziáky Z, Zengin G (2019) Investigation of Chemical Profile, Biological Properties of Lotus corniculatus L. Extracts and their Apoptotic Autophagic Effects on Breast Cancer Cells. J Pharm Biomed Anal 174:286–299. https://doi.org/10.1016/j.jpba.2019.05.068 Lee J, Jang H, Bak Y, Shin JW, Jin H, Kim YI, Ryu HW, Oh SR, Yoon DY (2019) Calotropis gigantea Extract Induces Apoptosis through Extrinsic/Intrinsic Pathways and Upregulation of Reactive Oxygen Species Generation in A549 and NCI-H1299 Non-small Cell Lung Cancer Cells. BMC Complement Altern Med 19:134. https://doi.org/10.1186/s12906-019-2561-1 Samarghandian S, Azimi-Nezhad M, Farkhondeh T (2019) Thymoquinone Induced Antitumor and Apoptosis in Human Lung Adenocarcinoma Cells. J Cell Physiol 234(7):10421–10431. https://doi.org/10.1002/jcp.27710 Additional Declarations The authors declare no competing interests. Supplementary Files HumairaGraphicalAbstract.tif Graphical Abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5897484","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":406801548,"identity":"af866f7c-969e-482f-99c2-0a0402a48896","order_by":0,"name":"Shehla Adhami","email":"","orcid":"https://orcid.org/0000-0001-6260-9311","institution":"Jamia Hamdard","correspondingAuthor":false,"prefix":"","firstName":"Shehla","middleName":"","lastName":"Adhami","suffix":""},{"id":406802338,"identity":"6a791f68-16a2-416d-b2cb-8f78169c6710","order_by":1,"name":"Humaira Farooqi","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-8222-7705","institution":"Jamia Hamdard","correspondingAuthor":true,"prefix":"","firstName":"Humaira","middleName":"","lastName":"Farooqi","suffix":""},{"id":406802339,"identity":"e5f0d151-4477-46eb-90d1-ce4603862043","order_by":2,"name":"Asrar Ahmad Malik","email":"","orcid":"https://orcid.org/0000-0003-1950-8747","institution":"Sharda University","correspondingAuthor":false,"prefix":"","firstName":"Asrar","middleName":"Ahmad","lastName":"Malik","suffix":""}],"badges":[],"createdAt":"2025-01-24 17:27:24","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5897484/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5897484/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74996073,"identity":"b81e91ec-4a93-4f2e-97a7-413cffb3806d","added_by":"auto","created_at":"2025-01-29 08:46:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a \u0026amp; b). DNA Fragmentation Analysis Induced by Ethanolic Leaves (CCLE) and Roots (CCRE) Extracts from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlorophytum comosum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein A549 Cells\u003c/strong\u003e. Induction of DNA fragmentation in A549 cells observed post CCLE and CCRE treatment. A total of 8 x 105 cells were exposed at (40 µg/ml and 80 µg/ml) for 24 hrs. DNA was electrophoresed on 1.8% agarose gel and stained with ethidium bromide. \u003cstrong\u003eFig 1(a)\u003c/strong\u003e CCLE induced dose dependent DNA fragmentation pattern in A549 cell line. \u003cstrong\u003eLane 1\u003c/strong\u003e- DNA marker ladder (1500 bp), \u003cstrong\u003eLane 2\u003c/strong\u003e- 80µg/ml, \u003cstrong\u003eLane 3\u003c/strong\u003e- 40 µg/ml, \u003cstrong\u003eLane 4\u003c/strong\u003e- negative control (untreated A549 cells), \u003cstrong\u003eLane 5\u003c/strong\u003e- positive control H2O2 \u003cstrong\u003e, Fig. 1(b)\u003c/strong\u003e CCRE treated cell line showing moderate DNA fragmentation pattern.\u003cstrong\u003e Lane 1\u003c/strong\u003e- negative control (untreated A549 cells),\u0026nbsp; \u003cstrong\u003eLane 2\u003c/strong\u003e- positive control H2O2, \u003cstrong\u003eLane 3\u003c/strong\u003e- 40 µg/ml, \u003cstrong\u003eLane 4\u003c/strong\u003e- 80µg/ml, \u003cstrong\u003eLane 5\u003c/strong\u003e- DNA marker ladder (1500 bp).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5897484/v1/d27d49baefea26a2efd9c911.jpg"},{"id":74995729,"identity":"1a4c85b1-7a7f-4647-b0e6-8bd3a3059cc5","added_by":"auto","created_at":"2025-01-29 08:38:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":135005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u0026nbsp; \u0026nbsp;Apoptosis Detection in Ethanolic Leaves (CCLE) and Roots (CCRE) Extracts Treated A549 Cells using PI-Annexin-V/FITC Staining by Flow Cytometry. \u003c/strong\u003eApoptosis induction was confirmed in A549 cell line by \u003cem\u003eChlorophytum comosum\u003c/em\u003e roots (CCRE) and leaves (CCLE) extracts after 24 hrs of exposure using Annexin V-FITC/PI staining. Flow cytometry analysis representing four quadrants indicating different stages of apoptosis. \u003cstrong\u003eUpper Left (UL)\u003c/strong\u003e- Dead Necrotic Cells, \u003cstrong\u003eUpper Right [A+/PI+]\u003c/strong\u003e- Late Apoptotic Cells, \u003cstrong\u003eLower Left (LL)\u003c/strong\u003e- Viable Cells \u003cstrong\u003e[A-/PI-], Lower Right (LR)\u003c/strong\u003e \u003cstrong\u003e[A+/PI-]\u003c/strong\u003e-Early Apoptotic Cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u0026nbsp;FACS Analysis of Apoptosis Detection in A549 Cells. \u003c/strong\u003eBar diagram representing percent apoptosis at different stages with dose dependent decrease in cell viability. Data is statistically significant at \u003cem\u003ep \u0026lt; 0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5897484/v1/f9477052fb68da34a354e33d.jpg"},{"id":74995727,"identity":"f39b8627-09a1-4eb3-98cf-06aab3d094b9","added_by":"auto","created_at":"2025-01-29 08:38:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. Cell Cycle Progression Analysis of Treated A549 versus Untreated A549 Cancer Cells by Florescence Activated Cell Sorter Flow Cytometry. \u003c/strong\u003eCell cycle analysis of A549 lung cancer cell line treated with various concentrations (40 µg/ml and 80 µg/ml) of CCLE and CCRE for 24 hrs using Propidium Iodide dye. Arrest of cells were observed with respect to untreated control A549 cells at S phase. Colchicine (25µM) was used as positive control. The data is represented in terms of percentage arrest. Data was statistically significant with \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and was calculated using one way analysis of variance (ANOVA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e \u0026nbsp;Percentage of A549 cells at various phases of cell cycle post treatment with \u003cem\u003eChlorophytum comosum\u003c/em\u003e leaves ethanolic (CCLE) and roots ethanolic (CCRE) extracts.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5897484/v1/42e861d55b5465de9d3a1180.jpg"},{"id":74996074,"identity":"8890328d-daa6-4f0c-a5bf-7e832faa972c","added_by":"auto","created_at":"2025-01-29 08:46:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1490112,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5897484/v1/c00814e3-016d-47d9-bd25-d194336e7dae.pdf"},{"id":74995726,"identity":"b03554f9-127f-47e5-979a-e131fd598115","added_by":"auto","created_at":"2025-01-29 08:38:50","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":799402,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"HumairaGraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-5897484/v1/e78877d150adb00877b3e2e0.tif"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eApoptosis Induction and S-Phase Cell Cycle Blockade in Human Lung Adenocarcinoma Cell Line (A549) by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlorophytum comosum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(Thunb.) Jaques\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLung cancer is the second most aggressive type of cancer contributing highest (21%) of all cancer deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Unlike other cancer types, where often the risk of cancer occurrence is influenced by genetic and epigenetic factors; lung cancer in majority, is a detrimental consequence of smoking habits observed among the human population and continued direct exposure of the harmful carcinogens present in the environment. Lung cancer incidence and mortality rates are tightly linked to cigarette smoking patterns [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Another challenge associated with lung cancer is its delayed detection mostly at advanced stage. Approximately 44% of people have advanced stage disease at diagnosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to American Cancer Society, in US alone, approximately 101,300 (81%) of the 125,070 lung cancer deaths in 2024 will be caused by direct cigarette smoking with an additional mortality of 3500 which will be caused by second hand smoke [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite the availability of modern chemotherapeutic interventions like personalized immunotherapy, chemotherapy, radiotherapy and surgical methods, the 5-year relative survival rate for all stages combined in lung cancer is 25% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The side effects of lung cancer treatment by these approaches render patients to look for alternative therapies that may help in improving the quality of life and naturally delaying the recurrent episodes of severe side effects as the disease progresses. Several forms of complementary and alternative medicine practiced under different traditional medicinal systems are now under investigation for its potential role in curbing and combating cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In China, Traditional Chinese Medicine (TCM) has been widely accepted as a mainstream form of complementary and alternative therapy for cancer patients [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A large number of studies on Traditional Chinese Medicine are elucidating the role of the herbs at the molecular level to understand its impact on cancer signaling pathways [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eChlorophytum comosum\u003c/em\u003e (Thunb.) Jaques belongs to family Lilliaceae and genus \u003cem\u003eChlorophytum\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It is commonly known as Spider plant and is worldwide popular for its ornamental value. In Traditional Chinese Medicine, it is used for the treatment of fractures, burns and respiratory ailments such as bronchitis and asthma [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In India, roots of \u003cem\u003eChlorophytum comosum\u003c/em\u003e are used as a substitute for another medicinal \u003cem\u003eChlorophytum\u003c/em\u003e species (\u003cem\u003eChlorophytum borivilianum\u003c/em\u003e) for preparation of an important class of ayurvedic drugs known as Rasayna [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Besides, several other species belonging to genus \u003cem\u003eChlorophytum\u003c/em\u003e are well known for their pharmacological properties [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The genus \u003cem\u003eChlorophytum\u003c/em\u003e is predominantly rich in saponins and has been extensively studied for its cytotoxic properties [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, as far as \u003cem\u003eChlorophytum comosum\u003c/em\u003e is concerned not much work has been done to discover its potential as an anticancer herb. Preliminary findings from earlier studies indicates its cytotoxic potential against selected cancer cell lines, however, these studies were only limited to initial cytotoxicity screening without deciphering the mechanism of action [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Saponins isolated from roots of \u003cem\u003eChlorophytum comosum\u003c/em\u003e have been found to exhibit cytotoxic and anti-tumour promoter activity in HeLa cancer cell line [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In an another important study, DNA damage in four different human cancer cell lines viz. HeLa, CCRF-HSB-2, HL-60 and U937 cells under the effects of n-butanol root fraction from \u003cem\u003eChlorophytum comosum\u003c/em\u003e was demonstrated using qualitative DNA fragmentation assay and the Terminal Deoxynucleotidyl Transferase (TdT) mediated biotin dUTP Nick End Labeling (TUNEL) method [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In a study, methanolic leaf extract of \u003cem\u003eChlorophytum comosum\u003c/em\u003e was evaluated for its chemical profile and its different sub fractions were explored for the bioactivity. The study revealed steroids and isoprenoid as the most abundant compounds present in methanolic leaf extract with higher percentage of unsaturated fatty acids and neophytadiene as the second highest abundant compound. Biological assessment of its sub fractions viz. chloroform, n-hexane, n-butanol and water revealed significant \u003cem\u003ein vitro\u003c/em\u003e antioxidant and selective cytotoxic activity against HeLa cancer cell lines [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recently, progressive neuroprotective efficacy of roots and leaves extracts of \u003cem\u003eChlorophytum comosum\u003c/em\u003e at the concentrations of 60 \u0026micro;g/ml and 90 \u0026micro;g/ml were demonstrated against glutamate excitotoxicity in a culture of rat cerebellar neurons retrieved from 7\u0026ndash;9 day old rats [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. \u003cem\u003eChlorophytum comosum\u003c/em\u003e inspite of having a strong Ethnopharmacological base and as an important ingredient in nutritional/health supplement in different traditional medicinal systems such as Traditional Chinese Medicine (TCM), African Traditional Medicine (ATM) and Ayurveda is yet unexplored on scientific grounds for its therapeutic potential [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, with view of its traditional usage for treatment of respiratory ailments, so far, no biological activity has been reported to explore its antiproliferative efficacy against lung cancer. Thus, keeping in view of the earlier literature and Ethanomedical lead, the present study aimed to further investigate the anticancer potential of ethanolic roots and leaves extract of \u003cem\u003eChlorophytum comosum\u003c/em\u003e against human lung adenocarcinoma cell line (A549) by understanding the mode of cancer cell death prompted by the extracts.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and Reagents\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSolvents used in the study i.e. n-Butanol, Petroleum Ether, Water, were of HPLC/LC-MS grade and were purchased from Merck (Darmstadt, Germany). Ethanol (China grade) and Hydrogen peroxide solution was purchased from local commercial supply (Thomas Baker, India). Cell culture media DMEM with phenol red (#1932403), RPMI-1640 (# 1898961), Fetal Bovine Serum (#10438034), Trypsin\u0026ndash;EDTA solution with phenol red (#1897336) and antibiotic solution PenStrep (#192493) were purchased from Gibco USA. MTT reagent (#MICB8173V), Propidium iodide (# P4864), RNase A, Standard Doxorubicin and Colchicine were obtained from Sigma Aldrich USA. FITC-Annexin V apoptosis detection kit was obtained from Invitrogen USA (# V-13242). Standard Vinblastine sulphate was purchased from Cipla (India). Sheath buffer was purchased from BD Biosciences-IN. All the cell lines used in the study were procured from ATCC (USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Plant Material Collection\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMature roots and leaves part of \u003cem\u003eChlorophytum comosum\u003c/em\u003e; cultivated for a period of 180 days were harvested in June 2016 from Herbal Garden, Jamia Hamdard, New Delhi. The plant parts collected were healthy and disease free. The plant samples were identified by the botanist Dr. Sunita Garg (NISCAIR, New Delhi) and the voucher specimens bearing no. NISCAIR/RHMD/CONSULT/ 2016/2975-02 were deposited in the herbarium.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Drug Preparation and Treatment for MTT Assay\u003c/h2\u003e \u003cp\u003eFor MTT assay, the semi dried extracts were weighed and initially prepared in DMSO with a final concentration of DMSO\u0026thinsp;\u0026lt;\u0026thinsp;0.1%. The samples were two fold serially diluted to 6 different concentrations (10\u0026ndash;320 \u0026micro;g/ml) [Stock conc. 3.2 mg/ml; Working conc. 0.32 mg/ml] using incomplete DMEM medium. Vinblastine sulphate served as positive control with 6 different test concentrations (3.12\u0026ndash;100 \u0026micro;M) [Stock conc.1.2 mM] [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Cells with 1% DMSO served as vehicle control, while, cells with only media served as negative control. Time duration for treatment was 24 hrs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell Lines Maintenance\u003c/h2\u003e \u003cp\u003eHuman lung adenocarcinoma cell line (A549) [CCL-185] and human normal lung cell line (L-132) [CCL-5] were procured from the American Type Culture Collection (Manassas, VA, USA). The cell lines obtained were pre- authenticated (molecularly characterized). Cell lines quality control specifications and authentication documentation were confirmed from the available information on the ATCC website as reference. Cell lines were maintained in DMEM/RPMI-1640 medium supplemented with 10% heat inactivated FBS and 1% penicillin/streptomycin solution with 5% CO\u003csub\u003e2\u003c/sub\u003e supply at 37\u0026deg; C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Experimental\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Preparation of Plant Extracts\u003c/h2\u003e \u003cp\u003eEthanolic extracts of roots (CCRE) and leaves (CCLE) of \u003cem\u003eChlorophytum comosum\u003c/em\u003e were prepared using hot continuous Soxhlet extraction method. Briefly, fresh plant materials were collected and washed to remove debris, shade dried under sunlight for seven consecutive days to remove the moisture content until no change in weight was observed. The grinded plant material (10 gm) each was defatted using petroleum ether (1:15 w/v), dried and suspended in ethanol (150 mL), and extracted using Soxhlet apparatus for 24 hrs at 50\u003csup\u003e0\u003c/sup\u003e C. The extracts were then collected, filtered, and partitioned using n-butanol until the upper layer became colorless. The upper layer was further collected, pooled and concentrated on a rotary evaporator at 55\u003csup\u003e0\u003c/sup\u003e C and 55 mbar pressure until the semi-dried substance was obtained. The semi-dried substance was further lyophilized to remove the traces of the solvent. The samples were then stored in airtight vials at 4\u003csup\u003e0\u003c/sup\u003e C till further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Cell Cytotoxicity Assay by MTT\u003c/h2\u003e \u003cp\u003eRepresenting methodology from our previous data for continuation of the study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], the cytotoxic activity in terms of % cell inhibition by roots ethanolic (CCRE) and leaves ethanolic (CCLE) extracts were measured using cell based colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction MTT assay with the treatment time of 24 hrs [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, monolayer cell culture (A549 \u0026amp; L-132) grown in T\u003csub\u003e25\u003c/sub\u003e cell culture flask was trypsanized using trypsin EDTA solution with phenol red and collected as pellet after centrifugation at 1000 rpm in 1 ml of complete medium. The total number of viable cells per ml was then counted with the help of Trypan blue dye (1:1 ratio with dilution factor 2) using hemocytometer. The cell count was adjusted to 40,000 cells/well, and to each well of the 96 well microtiter plate, 100 \u0026micro;l of the diluted cell suspension containing 40,000 cells was added. After 24 hrs, media was removed, cells were washed once with fresh medium and further treated with 100 \u0026micro;l of varying concentrations of extracts (10\u0026ndash;320 \u0026micro;g/ml) and Vinblastine (3.12\u0026ndash;100 \u0026micro;M) for 24 hrs. Subsequently, post treatment, 10 \u0026micro;L of MTT dye (0.5 mg/mL) was loaded inside each well and incubated further for 4 hrs at 37\u003csup\u003e0\u003c/sup\u003e C in an incubator supplied with 5% CO\u003csub\u003e2\u003c/sub\u003e. MTT solution was removed and crystals formed were then dissolved in DMSO. Finally, absorbance was taken at 570 nm using microplate reader (Spectra Max, USA). The % cell inhibition was measured by using the following formula (Eq A.1). The concentration at which the test drug inhibited cell growth by 50% i.e. inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) was generated from the dose-response curves by nonlinear regression analysis using Graph Pad Prism software (8.1).\u003c/p\u003e \u003cp\u003e% Cell Inhibition by CCLE /CCRE extracts = [(C \u0026ndash; S) X 100] / C \u0026hellip;\u0026hellip;\u0026hellip;. (Eq A.1)\u003c/p\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;Absorbance of control; S\u0026thinsp;=\u0026thinsp;Absorbance of samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Determination of Test Doses Based on IC\u003csub\u003e50\u003c/sub\u003e Values\u003c/h2\u003e \u003cp\u003eThe half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process like cell growth by half. To determine the apoptosis inducing effects of \u003cem\u003eChlorophytum comosum\u003c/em\u003e leaves ethanolic extract (CCLE) and roots ethanolic extract (CCRE), the active lower and higher doses were set corresponding to the determined IC\u003csub\u003e50\u003c/sub\u003e values of each extract from our previously published data [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The doses ranges selected for CCRE with an IC\u003csub\u003e50\u003c/sub\u003e value of 68.68 \u0026micro;g/ml and CCLE with an IC\u003csub\u003e50\u003c/sub\u003e value of 61.19 \u0026micro;g/ml were 40 \u0026micro;g/ml \u0026amp; 80 \u0026micro;g/ml respectively. Both the IC\u003csub\u003e50\u003c/sub\u003e values were falling between these two upper and lower doses selected for the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 DNA Fragmentation Assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eQualitative estimation of apoptosis inducing effects of the \u003cem\u003eChlorophytum comosum\u003c/em\u003e ethanolic extracts were measured using DNA fragmentation ladder assay. The formation of distinct DNA fragments of oligonucleosomal size (180\u0026ndash;200 bp length) is a biochemical hallmark of apoptosis in many cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These DNA fragments can be extracted from the cells post treatment and visualized using agarose gel electrophoresis method. Briefly, A549 cells seeded in DMEM medium at a concentration of 8 x 10\u003csup\u003e5\u003c/sup\u003e cells per 35 mm dish, incubated at 37\u003csup\u003eo\u003c/sup\u003e C, 5% CO\u003csub\u003e2\u003c/sub\u003e were treated with two concentrations i.e. (40 \u0026micro;g/ml and 80 \u0026micro;g/ml ) of each sample (CCRE \u0026amp; CCLE) for 24 hrs. Hydrogen peroxide served as control [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Total genomic DNA was taken out and DNA damage was evaluated using 1% agarose gel [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. DNA ladder of 1500 bp served as marker ladder to determine the apoptotic fragments.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.5 Cell Cycle Analysis by Propidium Iodide\u003c/h2\u003e \u003cp\u003eCell cycle analysis was performed by PI (Propidium iodide) staining method using flow cytometry [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, A549 cells (8 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml) cultured using 2 ml of DMEM medium in 6 well plates were treated with desired concentrations (40 \u0026micro;g/ml and 80 \u0026micro;g/ml) of test extracts (CCLE \u0026amp; CCRE) for 24 hrs. Post incubation, the medium was discarded and cells were gently scrapped using BD cell scrapper, transferred to RIA vials and centrifuged at 4500 rpm for 5 min at 4\u003csup\u003e0\u003c/sup\u003eC. The supernatant was discarded carefully retaining the cell pellets. Cell pellets were further washed twice using 1X PBS and fixed by resuspending in 300 \u0026micro;l of Sheath fluid followed by addition of 1mL of chilled 70% ethyl alcohol drop by drop with continuous gentle shaking and another 1 mL of chilled 70% ethyl alcohol added at once. The samples were stored at 4\u003csup\u003e0\u003c/sup\u003e C. Subsequently, cell pellets were washed twice with 2 ml of ice cold 1X PBS and then suspended in 450 \u0026micro;l of sheath fluid containing 16 \u0026micro;l PI [Stock conc. 0.05 mg/ml] and 16 \u0026micro;l RNaseA [Stock conc. 2 mg/ml; Working conc. 1 mg/ml] and incubated for 30 min in dark. The percent DNA content in various cycle phases in treated versus negative (untreated) control cells were determined using FACS Caliber instrument (BD Biosciences) with Cell Quest software (BD Biosciences, San Jose, CA, USA). Colchicine treated A549 cells (25 \u0026micro;M) served as positive control [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.6 Apoptosis Detection Using PI/Annexin V-FITC Staining\u003c/h2\u003e \u003cp\u003eApoptosis detection was performed using a commercial kit (Invitrogen V-3242) following the manufacturer's recommendations. Briefly, 8 x 10\u003csup\u003e5\u003c/sup\u003e A549 cells/ml plated in DMEM medium were treated with 40 and 80 \u0026micro;g/ml of CCLE and CCRE extracts for 24 hrs. The cells were then harvested, centrifuged at 4500 rpm for 5 min at 4\u003csup\u003e0\u003c/sup\u003e C and washed twice with ice-cold 1X PBS. The pellets were suspended with the 1X binding buffer (100 \u0026micro;L) and then PI (10 \u0026micro;L) [Stock conc. 1mg/ml] and Annexin V-FITC (3 \u0026micro;L) was added. The suspension was incubated in dark for 10 min at room temperature (RT). Before flow cytometry analysis, 100\u0026micro;L of 1X binding buffer was further added to the suspension. Data from 10,000 cells per sample were collected and analyzed by flow cytometry using a FACS Caliber instrument with Cell Quest software (BD Biosciences, San Jose, CA, USA). The results were compared with the untreated negative control A549 cells. Doxorubicin treated A549 cells (25 \u0026micro;M) served as positive control [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.7 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was performed with one way analysis of variance (ANOVA) to calculate the statistical significance with \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e as considered significant. The inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values were calculated by nonlinear regression curve (log inhibitor vs. normalized response variable slope) with the use of Graph pad Prism version 8.1 for Windows (Graph pad Prism Software, San Diego, CA, USA). All results were expressed as mean\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Selective Cytotoxicity of \u003cem\u003eChlorophytum comosum\u003c/em\u003e against Human Lung Adenocarcinoma (A549) Cell Line by MTT Assay\u003c/h2\u003e \u003cp\u003eBased on our earlier published data [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] on antiproliferative effects of \u003cem\u003eChlorophytum comosum\u003c/em\u003e, dose dependent selective cytotoxic activity towards A549 lung cancer cell line with an IC\u003csub\u003e50\u003c/sub\u003e values of 68.68 \u0026micro;g/mL and 61.19 \u0026micro;g/mL were demonstrated by both roots ethanolic (CCRE) and leaves ethanolic (CCLE) extracts respectively \u003cb\u003e(Table\u0026nbsp;1)\u003c/b\u003e. Leaves extract showed better cytotoxic response as compared to roots extract with lesser IC\u003csub\u003e50\u003c/sub\u003e values. In positive control taken as Vinblastine sulphate, maximum inhibitory effect with an IC\u003csub\u003e50\u003c/sub\u003e value of 18.79 \u0026micro;M and 20.71 \u0026micro;M was observed in A549 and L-132 respectively [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. While in case of normal human lung cell line (L-132), the IC\u003csub\u003e50\u003c/sub\u003e values were not calculated due to lesser percentage inhibition i.e.\u0026lt; 50%, indicating non-significant activity. For the present study, the test doses were selected as two active concentrations i.e. 40 \u0026micro;g/ml and 80 \u0026micro;g/ml for both roots ethanolic and leaves ethanolic extracts representing the lower and upper ranges between the determined IC\u003csub\u003e50\u003c/sub\u003e values to carry out the further investigations.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2\u003c/b\u003e \u003cb\u003eChlorophytum comosum\u003c/b\u003e \u003cb\u003eLeaves Ethanolic (CCLE) and Roots Ethanolic (CCRE) Extracts Promoted Nuclear DNA Fragmentation in A549 Cell Line\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOne major characteristic feature of apoptotic mode of cell death is the extensive morphological and biochemical changes that ensure the vanishment of dying cells without affecting and harming the other neighboring cells. In contrast, during necrosis mode of cell death, the cell gets destroyed leaving the traces of cellular contents that activate the chain reaction of inflammation in the cellular microenvironment. Formation of short distinct fragments of DNA of 180\u0026ndash;200 bp length visualized on agarose gel represents DNA damage and is considered as a biochemical hallmark of apoptosis process [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The apoptosis induction in A549 human lung adenocarcinoma cell line by the roots ethanolic and leaves ethanolic extracts of \u003cem\u003eChlorophytum comosum\u003c/em\u003e was qualitatively reported using gel based DNA fragmentation assay. A dose dependent internucleosomal fragmentation pattern was observed in A549 cells on 1.8% agarose gel stained with ethidium bromide when treated with CCLE extract at the concentrations of 40 \u0026micro;g/ml and 80 \u0026micro;g/ml for 24 hrs. Fine smearing throughout the gel represented significant DNA damage. Similarly in case of CCRE extract, significant DNA damage was observed at both doses, however, compared to leaves (CCLE) extract, the damage in roots extract was moderately recorded at 80 \u0026micro;g/ml. Hydrogen peroxide taken as positive control was used to compare the fragmentation pattern. Overall, these results suggested that both extracts induced DNA fragmentation in A549 cell line indicating DNA damage by apoptosis \u003cb\u003eFig.\u0026nbsp;1 (a \u0026amp; b).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 \u003cem\u003eChlorophytum comosum\u003c/em\u003e Induced Early and Late Apoptotic Cell Death in A549 Cells\u003c/h2\u003e \u003cp\u003eThe apoptotic effect of \u003cem\u003eChlorophytum comosum\u003c/em\u003e roots ethanolic (CCRE) and leaves ethanolic (CCLE) extracts was confirmed using Annexin- FITC/PI staining by evaluating the stages of cell death being early apoptotic, late apoptotic and necrotic. Apoptosis initiation is markedly represented by the acquisition of surface changes by dying cells such as the expression of thrombospondin binding sites, loss of sialic acid residues and exposure of phospholipids like phosphatidylserine (PS) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Early apoptotic events start with the translocation and exposure of phosphatidylserine to the outer layer of the membrane with intact cell membrane. True apoptotic cells can be quantitated using Annexin V dye which preferentially binds to negatively charged phospholipids like PS in the presence of Ca\u003csup\u003e2+\u003c/sup\u003e; the events of which are recorded in the form of fluorescence signal by flow cytometry. Staining cells simultaneously with FITC-Annexin V and the Propidium iodide allows the discrimination of intact cells by early apoptotic, late apoptotic or necrotic cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Apoptosis induction studies using Annexin V-FITC/PI staining demonstrated the occurrence of apoptosis in A549 cells after treatment with CCLE and CCRE extracts for 24 hrs. Dose dependent decrease in cell viability with varying % of cells in early apoptotic, late apoptotic and necrosis stages were recorded \u003cb\u003eFig.\u0026nbsp;2a.\u003c/b\u003e The 40 and 80 \u0026micro;g/ml treatment of leaves extract (CCLE) induced early apoptosis by 5.69%, 1.95%, and late apoptosis by 10.19%, 7.21% respectively. The roots extract (CCRE) treatment at 40 \u0026micro;g/ml and 80 \u0026micro;g/ml has induced early apoptosis by 9.67%, 5.38% and late apoptosis by 3.98%, 8.15% respectively. Necrotic cells were found to be 13.71%, 30.32% and 4.77%, 16.93% at 40 \u0026micro;g/ml and 80 \u0026micro;g/ml in CCLE and CCRE extracts respectively \u003cb\u003eFig.\u0026nbsp;2b.\u003c/b\u003e In control untreated A549 cells, 91% of viable cell remained intact with 8.32% early apoptotic cells with negligible late apoptotic cells i.e. 0.21% and no necrotic detected cells \u003cb\u003eFig.\u0026nbsp;2b.\u003c/b\u003e These results indicate that the death of A549 cells induced by \u003cem\u003eChlorophytum comosum\u003c/em\u003e was mediated by apoptosis. Doxorubicin used as positive control (standard), detected 14.44% early apoptotic, 30.66% late apoptotic, and 12.21% necrotic cells with 42.69% of viable cells \u003cb\u003eFig.\u0026nbsp;2b.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cem\u003eChlorophytum comosum\u003c/em\u003e Arrested Cell Cycle Progression at S Phase\u003c/h2\u003e \u003cp\u003eThe effects of leaves ethanolic (CCLE) and roots ethanolic (CCRE) extracts of \u003cem\u003eChlorophytum comosum\u003c/em\u003e were seen on cell cycle progression analyzed using PI staining detected by flow cytometry method. The treatment of A549 cells with CCLE and CCRE extracts for 24 hr at two different dosages (40 \u0026micro;g/ml and 80 \u0026micro;g/ml) led to the cell cycle arrest at S phase \u003cb\u003eFig.\u0026nbsp;3a.\u003c/b\u003e The cell cycle arrest was found to be in dose dependent manner with two fold change which was observed in both the cases from lower to higher concentrations, however, the leaves extract was found to be most effective in accumulating and arresting cells at S phase followed by the roots extract. CCLE extract has shown dose dependent S phase arrest of 23.23% and 42.86% at 40 \u0026micro;g/ml and 80 \u0026micro;g/ml respectively, compared to control untreated cells which showed only 5.90% arrest in A549 cells at S phase. While in CCRE extract, S phase arrest was 19.64% at 40 \u0026micro;g/ml and 38.03% at 80 \u0026micro;g/ml. Standard Colchicine at 25 \u0026micro;M showed 51.33% S phase arrest and 5.08% G2 arrest in A549 cells \u003cb\u003eFig.\u0026nbsp;3b.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe exploration of anticancer drugs from plant source is rapidly evolving with various high ends \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein silico\u003c/em\u003e screening techniques which helps in the preliminary identification of the active target biomolecules that may serve as lead candidates in anticancer therapy [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Several phytochemicals from plants have been found to possess anticancer activity by virtue of their ability in modulating the cell growth regulatory mechanisms i.e. cell cycle regulation and by inducing apoptosis in cancer cells when treated [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The apoptosis inducing ability of the phytoconstituents in immortal cancer cells is thus, a remarkable attempt to control the progression and spread of the cancer. This is achieved by the manipulation and activation of various cancer signaling pathways at the molecular level [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the present study, an attempt has been made to evaluate the anticancer potential of ethanolic roots and leaves extracts from \u003cem\u003eChlorophytum comosum\u003c/em\u003e against A549 human non-small cell lung carcinoma cell line by determining the mode of cell death and its underlying mechanism. \u003cem\u003eChlorophytum comosum\u003c/em\u003e is reported to be used in treatment of various respiratory ailments in Traditional Chinese Medicine (TCM) and has been found to be useful in relief from bronchitis and asthma [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Saponins are the chief active constituents found in \u003cem\u003eChlorophytum comosum\u003c/em\u003e and possess cytotoxic potential against cancer cell lines [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, till date no scientific studies were carried out with regard to respiratory ailments that could also justify its ethnomedical importance. To our best of knowledge, our study for the first time evaluated the apoptosis inducing potential of \u003cem\u003eChlorophytum comosum\u003c/em\u003e against lung cancer using \u003cem\u003ein vitro\u003c/em\u003e human lung adenocarcinoma cell line (A549) model which is an extensively studied model for human lung epithelial cancer and possesses the high metastatic potential [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This work is in continuation of our previously published work, where cytotoxicity studies by MTT assay revealed significant antiproliferative effects by chemically characterized ethanolic roots and leaves extracts selectively towards A549 cell line. While, no antiproliferative effects of the extracts at the same tested concentrations were observed against normal human lung cell line L-132. On the other hand positive control Vinblastine exhibited significant cytotoxicity towards both A549 and L-132 cell lines. In the present study, based on the earlier demonstrated IC\u003csub\u003e50\u003c/sub\u003e values, two active concentrations of the extracts were selected and we further investigated the markers of cell death process to ascertain the bioactivity of \u003cem\u003eChlorophytum comosum.\u003c/em\u003e We observed a systematic mode of cell death via apoptosis in A549 cells under the effect of the treatments. The early markers in apoptosis induced cell death involve internucleosomal DNA damage, cell membrane disruption and expression of Phosphatidylserine on outer membrane surface [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In our study, all these changes were systematically evident in A549 lung cancer cells post treatment. Annexin V-FITC/PI flow cytometry studies revealed increase in early apoptosis, late apoptosis and necrotic cells after CCLE and CCRE extracts treatment for 24 hrs with dose dependent decrease in the cell viability. DNA fragmentation studies indicated the DNA damage initiated by the treatment in response to non-treated control cells where no such activity was observed. However, the potency of each extract exhibited varied response at different concentrations selected for the study. CCLE extract exhibited greater potential as compared to CCRE extract. Another aspect associated with cancer control and progression is cell stage specific growth during which many proteins are altered and leads to malignant conditions. Studies reveals that plant derived drugs block cell cycle progression at various stages of cell cycle such as G0/G1, S or G2/M phases to induce apoptotic cell death [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our study reveals the arrest of cell cycle of malignant A549 cells when treated with CCLE and CCRE extracts at S phase \u003cb\u003eFig.\u0026nbsp;3\u003c/b\u003e. The growth arrest and apoptosis inducing features of the plant extracts have been found significant in number of studies. In a study conducted by Yerlikaya et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], ethyl acetate extract from \u003cem\u003eLotus corniculatus\u003c/em\u003e exhibited significant apoptotic driven cell death via increased intranuclear influx of Ca2\u003csup\u003e+\u003c/sup\u003e in MDA-MBA-231 breast cancer cell line. In a similar findings by Lee et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], ethanolic extract from \u003cem\u003eCalotropis gigantean\u003c/em\u003e induced apoptosis through the activation of extrinsic and intrinsic pathways, cell cycle arrest, and ROS generation in A549 and NCI-H1299 lung cancer cell lines. In a study conducted by Samarghandian et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], antineoplastic potential of a plant derived Thymoquinone was assessed using flow cytometry studies on A549 cell line and were found to significantly up regulate the apoptotic modulators viz. \u003cem\u003ep53\u003c/em\u003e, caspase 3 and 9 with significantly elevated Bax/Bcl-2 ratio thereby, concluding initiation of apoptosis in A549 cells via caspase cascade dependent pathways. Thus, with insights from earlier literature and following the lead of our previous work where significant cytotoxic effects of ethanolic extracts of \u003cem\u003eChlorophytum comosum\u003c/em\u003e were evident on human lung carcinoma cell lines; the present study confirms the antiproliferative and antitumor effects of CCLE and CCRE extracts in A549 lung cancer cell line. This the first report of lung cancer cell toxicity and programmed cell death via apoptosis pathway in A549 cell line by \u003cem\u003eChlorophytum comosum\u003c/em\u003e ethanolic roots and leaves extracts. Such data indicates that \u003cem\u003eChlorophytum comosum\u003c/em\u003e have therapeutic phytochemical leads as apoptosis inducers in lung cancer cells that can be further isolated, characterized and studied and their potency as anticancer agents could be explored in the management of lung cancer. However, persuasive investigation is required to further characterize and elucidate the cancer signaling pathways involved in the bioactivity of active constituents from \u003cem\u003eChlorophytum comosum\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eActive constituents from roots and leaves parts of \u003cem\u003eChlorophytum comosum\u003c/em\u003e possess significant cytotoxic and apoptosis inducing potential against A549 lung cancer cell line suggestive of its anticancer potential. However, further in line research, based on \u003cem\u003ein vivo\u003c/em\u003e and clinical studies will reveal novel bioactive leads from \u003cem\u003eChlorophytum comosum\u003c/em\u003e (Thunb.) Jaques.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eChlorophytum comosum\u003c/em\u003e roots ethanolic extract\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCLE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eChlorophytum comosum\u003c/em\u003e leaves ethanolic extract\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal Bovine Serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate Buffer Saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eA549\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Lung Adenocarcinoma Cell Line\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFinancial Support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data that support the findings of this study are available from the corresponding author [Dr. Humaira Farooqi] upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Professor M.Z. Abdin (Department of Biotechnology, Jamia Hamdard) for providing facilities for sample preparations. Mr. Hashmat (Herbal Garden, Jamia Hamdard) for supervising and providing plant samples. Dr. Sunita Garg and Professor M.P. Sharma for authentication of plant samples and Hamdard National Foundation for granting research fellowship to Dr. Adhami. The kind guidance and technical assistance of Skanda Life Sciences, Bangalore is highly appreciated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report there are no competing interests to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN, Jemal A (2024) Cancer Statistics, 2024. 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BMC Complement Altern Med 19:134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12906-019-2561-1\u003c/span\u003e\u003cspan address=\"10.1186/s12906-019-2561-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamarghandian S, Azimi-Nezhad M, Farkhondeh T (2019) Thymoquinone Induced Antitumor and Apoptosis in Human Lung Adenocarcinoma Cells. J Cell Physiol 234(7):10421\u0026ndash;10431. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcp.27710\u003c/span\u003e\u003cspan address=\"10.1002/jcp.27710\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Jamia Hamdard","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Apoptosis, A549, Chlorophytum comosum, Lung Cancer, Spider Plant, Phytoterapeutics","lastPublishedDoi":"10.21203/rs.3.rs-5897484/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5897484/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe ethanolic roots (CCRE) and leaves (CCLE) extracts were interrogated for their apoptotic potential against human lung adenocarcinoma cell line (A549) using DNA fragmentation, Annexin V-FITC/PI staining apoptosis assay and cell cycle analysis using flow cytometry.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur results revealed significant DNA damage and apoptosis induced cell death in A549 cell line on treatment with active concentrations (40 \u0026micro;g/ml and 80 \u0026micro;g/ml) of the ethanolic extracts with S phase cell cycle arrest.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis is the first study demonstrating the apoptosis inducing potential of chemically characterized bioactive compounds present in ethanolic leaves and roots extracts from \u003cem\u003eChlorophytum comosum\u003c/em\u003e against non-small cell human lung adenocarcinoma cell line. The study concludes that \u003cem\u003eChlorophytum comosum\u003c/em\u003e can be a potential candidate for the natural bioactive compounds that can be isolated, characterized and clinically evaluated for the development of novel naturally derived anti-cancer drugs against lung cancer.\u003c/p\u003e","manuscriptTitle":"Apoptosis Induction and S-Phase Cell Cycle Blockade in Human Lung Adenocarcinoma Cell Line (A549) by Chlorophytum comosum (Thunb.) Jaques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-29 08:38:30","doi":"10.21203/rs.3.rs-5897484/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56f884cc-4d1b-47ee-ae9a-89f7ae31ebcd","owner":[],"postedDate":"January 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43385765,"name":"Natural Product Chemistry"},{"id":43385766,"name":"Cell Cycle \u0026 Proliferation"},{"id":43385767,"name":"Cell Survival and Cell Death"}],"tags":[],"updatedAt":"2025-01-29T08:38:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-29 08:38:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5897484","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5897484","identity":"rs-5897484","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}