Cytotoxic Activity, Cell Cycle Inhibition, and Apoptosis-Inducing Potential of Athyrium hohenackerianum (Lady Fern) with Its Phytochemical Profiling

In the present study, we investigated the cytotoxic effects of Athyrium hohenackerianum ethanolic extract (AHEE) on the proliferation of breast, lung, and colon cancer cells. The AHEE was tested for its effect on the progression of the cell cycle, followed by induction of apoptosis determination by flow cytometry. Real-time qRT-PCR was also utilized to observe the initiation of apoptosis. In addition, GC-MS was performed in order to identify the active phytochemicals present in the AHEE. A cytotoxic activity with an IC50 value of 123.90 μg/mL against HCT-116 colon cancer cells was exhibited by AHEE. Following propidium iodide staining, annexin-V/PI, and clonogenic assays, AHEE treatment results in cell arrest in the S phase, causing an increase in the early and late phases of apoptosis and displaying antiproliferative potential, respectively. The morphological alterations were further monitored using acridine orange/ethidium bromide (AO/EB) staining. When compared with the control cells, features of apoptotic cell death, including nuclear fragmentation, in the AHEE-treated cells were noticed. The apoptosis was also confirmed by detecting the increased expression of p53 and caspase-3 along with the downregulation of Bcl-2. GC-MS analysis revealed that trans-linalool oxide, loliolide, phytol, 4,8,12,16-tetramethylheptadecan-4-olide, and gamma-sitosterol were the major phytochemical constituents. Based on these findings, it can be suggested that AHEE causes cellular death via apoptosis, which should be further explored for the identification of active compounds responsible for these observed effects. Therefore, AHEE can be used in the pharmaceutical development of anticancer agents for cancer therapeutics.


Introduction
Around the globe, cancer-related deaths are increasing as the incidence of cancer continues to rise [1]. As per the World Health Organization (WHO), colorectal cancer, lung cancer, and breast cancer are the three most frequently diagnosed cancers globally and remain the foremost reason of cancer-related deaths throughout the world [2]. Conventional cancer treatments are still the most common form of treatment, despite being mainly unsuccessful and causing many deaths due to side effects. In contrast, developing new cancer treatments derived from natural sources with fewer side effects has become an exciting field of research [3]. Plants are regarded as a potential repository of novel

Collection of Plant Material and Extraction.
e collection of A. hohenackerianum whole plants was carried out in the South Gujarat region of Gujarat state, India, in September 2020. e voucher specimen (BVBRC042) was deposited at Bapalal Vaidya Botanical Garden, Department of Biosciences, Veer Narmad South Gujarat University, Surat, Gujarat, India. e collected plant material was washed with tap water, and then, deionized water was used to rinse. ereafter, it was dried under shade at room temperature for 7 days. Upon drying, the samples were ground into powder and were then passed through a sieve that had a mesh size of 20 (sieve size 0.85 mm). 25 g powder of the whole plant of A. hohenackerianum was macerated in 85% ethanol in an electric shaker at the room temperature for 6 h. After filtering through Whatman filter paper No. 1, the extract solution was evaporated in a water bath at 60°C for 2 h. A total of 3.40 g of the extract was recovered, which was brown in color, solid in appearance, and bristle in texture.

Cytotoxicity by MTT Assay.
e AHEE was tested against human lung (A549), breast (MCF-7), and colon (HCT-116) cancer cells. e National Centre for Cell Science (NCCS), Pune, India, provided cell lines for use in this study. Cancer cell lines were cultured in flasks (25 cm 2 ) with 10% fetal bovine serum (FBS) (MP Biomedicals, Germany) in Dulbecco's Modified Eagle's Medium (DMEM) (MP Biomedicals, Germany) and 10,000 U/mL penicillin and 5 mg/ mL streptomycin antibiotic solution (Hi-Media, India) at 37°C in a humidified atmosphere with 5% CO 2 . Upon reaching 80% confluence, the cells were seeded at a density of greater than 1 × 10 5 cells per well in 96-well plates and incubated in the same conditions as above. Trypan Blue (Hi-Media, India) (0.4%) was used to stain the cells, and a hemocytometer was used to determine the viability. Afterward, cells were treated with AHEE at different concentrations (1.56-200 μg/mL) for 48 h. e plate was removed from the incubator, and the media containing AHEE was aspirated.
en, 200 μL of medium containing 10% MTT reagent (MP Biomedicals, Germany) was added to each well to get a final concentration of 0.5 mg/mL, and the plates were incubated for a further 3 h at 37°C in a humidified atmosphere with 5% CO 2 . is was then followed by the removal of the medium and the addition of 100 μL DMSO (Merck, Germany) to dissolve the formazan crystals. Using the ELISA reader (EL10 A, Biobase, China), the absorbance of the amount of formazan crystal was measured at 570 nm and 630 nm. e percentage growth inhibition was calculated after subtracting the background and the blank, and the concentration of the test drug needed to inhibit cell growth by 50% (IC 50 ) was calculated from the dose-response curve for the respective cell line. As a positive control, cisplatin was used [22].

Cell Cycle Analysis.
e flow cytometer was used to evaluate the cell cycle using propidium iodide (PI) staining. In a six-well culture plate, HCT-116 cells were plated at 5 × 10 4 cells per 2 mL and incubated in a CO 2 incubator for 24 h at 37°C. After incubation, the spent medium was aspirated and washed with 1 mL of 1X PBS. Cells were treated with AHEE (IC 50 ) concentration in 2 mL of the culture medium and further incubated for 24 h. One well was left blank as untreated, which was considered as a negative control. e floating and attached cells were collected and washed with chilled PBS. Following permeabilization, the cells were fixed for 1 h at 4°C in ice cold 70% ethanol. A staining solution (50 μg/mL PI and 20 μg/mL RNase A in PBS) was then added to the cells for 15 min at 37°C. Samples were mixed well and analyzed with the Cytomics FC500 Flow cytometer, Beckman Coulter, USA [23].

Fluorescent Double Staining with Acridine Orange/ Ethidium Bromide (AO/EB).
e cells were plated at a density of 3 × 10 5 cells/2 mL in 6-well plates and incubated at 37°C for 24 h in a CO 2 incubator. After aspirating the spent medium, 1 mL of PBS was added. e cells were treated with AHEE (IC 50 ) and further incubated for 24 h. For the second time, the medium was removed after incubation and washed in cold PBS. After that, the cells were suspended in 500 μL of AO/EB staining solution (10 μL of acridine orange (2 mg/mL) and 10 μL of ethidium bromide (2 mg/mL) in 1 mL of PBS), mixed thoroughly, and then incubated for 5 min. In the end, cells were washed with PBS bovine serum albumin three times, and images were taken immediately under a fluorescent microscope (XDFL series, Sunny Instruments, China) [24].

Colony Formation Assay.
In order to determine the antineoplastic effects on in vitro cell proliferation, the clonogenic assay was performed as described previously with  minor modifications [25]. In brief, the cells were cultured for 24 h in T-25 tissue culture flasks and exposed to AHEE (IC 50 ) for 24 h. Afterward, trypsinization was performed on the cells; the cells were counted and seeded into a 6-well plate (200 cells/2 mL medium per well) and cultured for 8 days.
Methanol was used to fix the colonies. Crystal violet (0.4 g/L) was used to stain the colonies, which were photographed, analyzed, and counted using ImageJ (v1. 48 (1) 2.6. Annexin-V Apoptosis Assay. A 6-well plate was seeded with HCT-116 cells at a density of 3 × 10 5 cells/2 mL and incubated in a CO 2 incubator at 37°C for 24 h. After removal of the spent medium, 1 mL of PBS was added. e cells were treated with AHEE (IC 50 ) in 2 mL of the culture medium and incubated for 12-16 h. An untreated well served as a negative control. At the end of incubation, the medium was removed from all the wells and transferred into 5 mL centrifuge tubes, which were then washed with 500 μL PBS. PBS was removed from the sample, and 200 μL of trypsin-EDTA solution was added and incubated for 3-4 min at 37°C. Afterward, the culture medium was transferred into the respective wells again and cells were harvested into the centrifuge tubes. e tubes were then centrifuged for 5 min at 300 × g at 25°C. e supernatant was then removed and washed twice with PBS. e PBS was completely removed, and cells were resuspended in 1X binding buffer at a concentration of 1 × 10 6 cells/ml. Following this, 100 μL of the solution (1 × 10 5 cells) was transferred to a 5 mL culture tube and 5 μL of AbFlour 488 Annexin V was added. e cells were gently vortexed and incubated for 15 min at 25°C in the dark. At the end, 2 μL of PI and 400 μL of 1X binding buffer were added to each tube and vortexed gently. Analyses of the samples were carried out by flow cytometry immediately after the addition of PI [26].

Gene Expression Analysis.
Tri-Pure Isolation Reagent (Sigma-Aldrich ® , India, 11667157001) was used to isolate the cellular RNA according to the manufacturer's instructions, and it was quantified with a nanodrop UV spectrophotometric analyzer (P 300, IMPLEN, USA). e RT-first strand synthesis kit (Qiagen, CA, USA, 330401) was used to reverse transcribe 1 μg of isolated RNA. SYBR green qRT-PCR (Applied Biosystems ® 7500 Fast Real-Time PCR machine, CA, USA) was used to measure the gene expression levels relative to control. e 2 −ΔΔCt method was used for the analysis, with values expressed as fold changes over the control value. Each primer pair (Table 1) was used separately. e following conditions were used to determine the relative gene expression: reverse transcription was performed at 45°C for 45 minutes as a starting point, initial denaturation at 95°C with 10 min hold, followed by 40 cycles of 95°C for 15 sec, and 60°C for 60 sec [28].

GC-MS Analysis of A. hohenackerianum Crude Extract.
Shimadzu Nexis GC-2030 equipped with a QP2020 NX mass spectrometer was used for the GC-MS analysis of AHEE. e helium was used as a carrier gas, which flowed at a rate of 1 mL/min. e GC-MS spectral detection method was based on electron ionization energy ionized at 70 eV, a scanning time of 0.2 s, and fragment masses in the range of 40 to 600 m/z. A volume of 1 μl and a temperature of 250°C were used for injection. Initially, a temperature of 50°C was set for 3 min in the column oven, then increased by 10°C per min to 280°C, and finally was set to 300°C for 10 min. A comparison of the phytochemicals present in the test samples with a library of authentic compounds maintained by the National Institute of Standards and Technology (NIST) revealed the presence of these compounds based on their mass spectral patterns, retention time (min), peak, area, and height [29].

Statistical Analysis.
All experiments were carried out in triplicate. Results are presented as the mean ± SD of the number of experiments performed. e significance of the results was determined among the treatments using one-way ANOVA followed by Tukey's post hoc test and Student's ttest at p < 0.05. e analyses were carried out using GraphPad Prism 5.0 software.

Cytotoxic Activity of A. hohenackerianum Crude Extract.
To determine the cytotoxic activity, various concentrations of AHEE (1.56-200 μg/mL) were applied to A549, MCF-7, and HCT-116 cancer cells and then the IC 50 values were determined. A dose-response inhibition curve was used to determine the values after 48 hours. Figure 2 illustrates the dose-response curve of exposure of cancer cell lines to the

Morphological Changes in HCT-116 Cells.
To explore the morphological alterations caused by AHEE, HCT-116 cells were monitored using AO/EB staining. As compared with the control cells, the features of apoptotic cell death, including nuclear fragmentation, in the AHEE-treated cells were noticed (Figure 4). ese results elucidated that the inhibition of AHEE on HCT-116 cell growth is linked with its induction of apoptosis.

Antiproliferative Potential of AHEE.
e AHEE was examined for its effect on colony-forming ability in HCT-116 cells. Using AHEE, clonogenic assays were conducted to test the differences between untreated and treated cells in terms of reproductive viability. e colonies formed after 8 days were compared between cancer cells seeded with and Evidence-Based Complementary and Alternative Medicine 5 without AHEE. A significant reduction in the number of colonies was observed in AHEE ( Figure 5).

Quantification of Apoptotic Cell Death of AHEE-Treated
Cells. e apoptosis induction by AHEE on HCT-116 cells was further confirmed using the Annexin-V and propidium iodide staining methods. e results of this experiment are shown in Figure 6. After treatment with IC 50 , there was a significant increase from 0.51 ± 0.30% to 9.52 ± 0.92% and 2.80 ± 0.38% to 22.3 ± 1.23% of early and late apoptotic/ necrotic cells, respectively.

Expression of Genes Related to Apoptosis.
Real-time PCR was used to detect the expression level of the apoptotic genes p53, Bcl-2, and caspase-3 in HCT-116 cells treated with AHEE. Compared to untreated cells, expression levels of caspase-3 and p53 were significantly increased, whereas the expression levels of Bcl-2 were significantly decreased in AHEE-treated cells (Figure 7).

Discussion
Medicinal plants have been the known source of medicines for the treatment of various ailments since ancient times. People around the globe have implemented the usage of botanicals for millennia. Although angiosperms and other higher plants have been extensively studied for their medicinal and economic value, ferns and fern-allies have been completely overlooked. ere is a strong emphasis on its use in Ayurvedic (Sushruta, Charka, and Samhita), Unani, Homeopathic, and other systems of medicine. People use them in the treatment of different kinds of illnesses like burns, colds, ascarid diseases, bleeding due to trauma, and diarrhea [30]. Preliminary screening on different sorts of bioactivities of traditional medicinal ferns has been reported [31][32][33][34][35][36]. However, no in-depth studies on any biological activities of these plants exist.
A. hohenackerianum contains a wide array of phytochemical constituents, making it one of the most valuable medicinal plants [37]. In this study, A. hohenackerianum was extracted using ethanol to obtain the largest amount of bioactive phytochemicals. Based on the results of this study, we found that the ethanolic extract of A. hohenackerianum conferred high cytotoxic activity on a variety of cancer cells, with good efficacy against human colon cancer cells HCT-116. In addition, we showed that the ethanolic extract of A. hohenackerianum arrests the cell cycle at the S phase and specifically induces apoptosis. is fact has been supported by numerous experiments, including labeling with Annexin V-/PI and activation of apoptosis signaling molecules. is study's findings are consistent with a prior study that found other species of Athyrium extracts have antiproliferative agents against several cancer cells. It has been reported that A. multidentatum shows cytotoxic activity against hepatocellular carcinoma (HepG2 cells) with IC 50 values of 220 μg/ mL and 114 μg/mL after 24 h and 48 h, respectively [38]. It was also reported that A. multidentatum crude extract causes cytotoxicity in HL-7702 liver cells with IC 50 values of 332 μg/ mL and 304 μg/mL after 24 h and 48 h, respectively [39]. e butenolide derivative Striatisporolide A of A. multidentatum exhibits potent cytotoxic activity against human lung cancer cells (A549) with an IC 50 of 7.75 μg/mL [39].
To investigate the mechanisms involved in the growth inhibition of HCT-116 cells treated with A. hohenackerianum, the cell cycle distribution was analyzed. In the present study, it was found that cells treated with A. hohenackerianum accumulated at the S phase of cells, and therefore, this suggests that HCT-116 cells undergo apoptosis since accumulation of cells at this phase is considered a biomarker for DNA damage as well as an indicator of cell death by apoptosis [40]. As part of the confirmation of apoptosis induction, the Annexin-V/PI apoptosis detection assay was used, which is widely used to distinguish both early and late stages of apoptosis [41]. e apoptotic cell death mode of A. hohenackerianum was confirmed by a shift toward early and late apoptotic cell populations, suggesting the extract exerted an apoptotic effect. e dual staining of Annexin-V/PI and the cells accumulated in the S phase, as well as the morphological changes that occurred during the cell death evident with AO/EB after treatment with A. hohenackerianum crude extract, strongly indicate that the cells are undergoing apoptosis. It is well known that intact plasma membrane integrity can be used as a major indicator of apoptotic cells morphologically [42,43]. In contrast to apoptosis, necrosis is characterized by a loss of integrity in the cell membrane [42]. e dual AO/EB fluorescent staining method is convenient for detecting changes in cell membranes caused by apoptosis. e AO binds to DNA in intact cells and emits green fluorescence. In damaged cells, EB penetrates the membrane and binds to the DNA and fluorescence of orange-red is emitted [44]. erefore, AO allows the staining of apoptotic cells as well as live cells, and EB permits the staining of both necrotic and late apoptotic cells.  Evidence-Based Complementary and Alternative Medicine Furthermore, we also performed a more prolonged clonogenic survival test and found that A. hohenackerianum inhibited the survival of HCT-116 cells for a longer period of time. As a result, it should be noted that the crude extract of A. hohenackerianum has the potential to be used as a treatment or management for colon cancer, since targeting a cell population that is clonogenic/tumor-initiating/stemlike is believed to be essential for success with cancer therapy. e apoptotic process is regulated by genes such as p53, caspase-3, and Bcl-2 [45]. A qRT-PCR technique was used to analyze the expression of these apoptotic genes to support the results from the flow cytometry. Compared to the control, treatment with the extracts significantly lowered the antiapoptotic Bcl-2 gene and significantly increased the apoptotic Bax and caspase-3 genes, indicating that A. hohenackerianum acts through caspase-dependent apoptotic pathways. It has been reported that a similar event occurred in many studies in which natural product extracts increased the expression of these genes in different human cancer cells, thus causing them to undergo apoptosis [46][47][48][49]. Overall, our study confirms that A. hohenackerianum induces apoptosis in HCT-116 cancer cells.   e medicinal properties of plant extracts are largely due to the secondary products that act in synergism rather than as a single compound [50][51][52]. GC/MS is a useful and dependable method for identifying complex plant extracts in an efficient and timely manner [53]. is study identified different classes of phytochemical constituents of A. hohenackerianum as having an inhibitory effect on colorectal cancer cells' growth [54], suggesting that these compounds may play a role in the observed activity against HCT-116 colon cancer cells.
e natural compound butyric acid is found in food. e anticancer activity of butyric acid has been reported against acute myeloid leukemia, Lewis lung carcinoma cells, and colorectal carcinoma cells [55]. Linalool showed the strongest activity against a broad range of cancers, such as carcinoma of the cervix, stomach, skin, lung, and bone with IC 50 ranging from 82.3 to 113.6 μg/mL [56]. Phytol is a substance found in chlorophyll and has been shown to be cytoprotective against oxidative stress. e anticancer and immune-enhancing properties of phytol are well-documented. By regulating macrophage function, phytol not only enhances natural killer cells that remove cancer cells but also strengthens immunity.
ere is evidence that phytol has an anticancer effect against breast, prostate, cervical, colorectal, lung, and skin cancers, with IC 50 values ranging from 15.51 to 69.67 μM [57]. Gamma-sitosterol is a compound that belongs to the stigmastanes and derivatives family of organic compounds. It has also anticancer effects against several types of cancer including, breast, lung, hepatocellular, and colorectal cancer [58]. erefore, it can be concluded that the anticancer effects seen in the extract could be due to the presence of these compounds.

Conclusion
e ethanolic extract of A. hohenackerianum inhibited the proliferation of various cancer cells. In particular, we demonstrated that the A. hohenackerianum extract inhibits the growth of HCT-116 colon cancer cells by apoptosis by inducing cell arrest in the S phase and causing dose-dependent rises in early and late apoptotic cell populations. Moreover, upregulation of apoptosis gene markers in HCT-116 cells confirmed the initiation of apoptosis by A. hohenackerianum. Based on preliminary findings, A. hohenackerianum has the potential to be a new natural source of anticolon cancer compound(s) that can trigger apoptotic cell death. However, despite the numerous advantages, toxicity concerns are always present with the variety of plants and can be unsafe for the sensitive populations. erefore, in vivo toxicological assessment is a mandatory requirement and must be done prior to the drug development, which is a limitation in this study. Furthermore, certain assays such as migration assay, angiogenesis assay, and protein expression by Western blotting can also be performed to confirm the efficacy of A. hohenackerianum and identify the possible cellular and molecular mechanisms involved in the anticancer activity.

Data Availability
All data generated or analyzed during this study are included in this article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.