Chemicals and reagents

Cell culture reagents such as Dulbecco's Modified Eagle Medium (DMEM: AT006), Minimum essential medium (MEM: AT154), and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT: TC191) were purchased from Hi-Media Laboratories (India). Streptomycin (91014), penicillin (40309), and gentamicin (37636) were purchased from (Sisco Research Laboratories Pvt. Ltd., India). All the chemicals used for phytochemical extraction and column chromatography were obtained from Merck (Germany). Antibodies against poly (ADP-ribose) polymerase (PARP: 9532S), caspase 3 (9662S), caspase 8 (4790S), caspase 9 (9508S), B-cell lymphoma-2 (BCL-2) homology 3-interacting-domain death agonist (BID: 2002T), phospho-AKT (4060T), p53 (9282S), BCL-2 associated X protein (BAX: 2772T), and apoptosis inducing factor (AIF: 5318P) were procured from Cell Signaling Technology (USA). β-actin (A2228), vinculin (V9131) and all other chemicals were purchased from Sigma (USA) if not otherwise indicated.

Cell lines and culture conditions

The human colorectal carcinoma cell line HCT116, colorectal adenocarcinomas HT-29 and Caco-2, human cervical cancer cell line HeLa, human pancreatic cancer cell line PANC-1, human malignant melanoma cell line A375, mouse fibroblast cell line L929 and human embryonic kidney cell line HEK-293 were procured from the National Centre for Cell Science, Pune, India and maintained in DMEM or MEM containing 10% fetal bovine serum (Gibco 10270) with 55 μg/mL gentamicin, 100 μg/mL streptomycin, 50 U/mL penicillin, and 2 μg/mL amphotericin B. The cells were grown at 37°C in a humidified atmosphere of 5% CO₂ (BB150, Thermo Scientific) and passaged over two times in a week and confirmed mycoplasma free on regular basis. Exponentially growing cells were taken for all the analysis.

Plant specimen

The plant specimen, P. vettiveroides were collected on 20th June 2019 from the greenhouse (Department of Biotechnology, University of Calicut, India). It was identified and authenticated by Dr. A. K. Pradeep, Curator, Department of Botany, University of Calicut, India. The voucher specimen with an accession number—CALI 7007 was deposited in the herbarium at the same department. The name of the plant has been checked with the “World Flora Online (WFO)” [18].

Leaf extract preparation and bioactive fraction (PVF) isolation

Organic solvent extracts of the leaf were prepared by successive polarity gradient extraction. Briefly, 25 g of dried leaf powder was treated successively with 500 mL of each solvent (n-hexane, ethyl acetate, and methanol) under room temperature with constant shaking for 24 h. The extracts obtained were dried using a vacuum rotary evaporator. The most cytotoxic extract (ethyl acetate), was subjected to column fractionation by conventional silica gel column chromatography (60–120 mesh, column size 25 cm × 3 cm) using an n-hexane: ethyl acetate solvent system. The crude ethyl acetate extract (500 mg) was loaded on top of the packed silica gel and the column was eluted stepwise with 100 mL of hexane and hexane: ethyl acetate solvent system (100:0), (95:5), (90:10), (85:15), (80:20), (75:25), (70:30), (65:35), (60:40), (55:45), (50:50), (45:55), (40:60), (35:65), (30:70), (25:75), (20:80), (15:85), (10:90), (5:95), (0:100).

The cytotoxic activity of the 82 fractions collected was tested and two cytotoxic fractions (fractions 21 and 22) were identified. As fraction 21 exhibited a better cytotoxic profile, it was considered for further analysis and was designated as PVF.

MTT assay

The cytotoxic potential of the leaf extracts and PVF was assessed using an MTT assay. Briefly, cells were seeded in 96-well plates (2 × 10³ cells/well) other than Caco-2 which was seeded (6 × 10³ cells/well) and treated with different concentrations of plant extracts and PVF for 72 h. The drug-containing media was then replaced with fresh media containing 1 mg/mL MTT per well and incubated for 2 h. At the end of incubation, 100 μL of lysis buffer (20% sodium dodecyl sulfate in 50% dimethylformamide) was added to the wells and incubated further for 30 min at 37°C. The optical density was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (BioTek, Synergy H1). The relative cell viability in percentage was calculated as [A570 (treated cells)/A570 (untreated cells)] × 100. The half-maximal inhibitory concentration (IC₅₀) values were extrapolated from polynomial regression analysis of experimental data.

Clonogenic assay

The proliferative propensity of the HCT116 cells after exposure to PVF was assessed by clonogenic assay. Briefly, 200 cells per well, seeded in a six-well plate were treated with different concentrations of PVF and curcumin (cur: 9 μg/mL) was used as a positive control. After 72 h, the drug-containing medium was replaced with fresh medium, and the plate was incubated for a week. The clones developed were fixed in glutaraldehyde (6%) and stained using crystal violet (0.5%) for 60 min. The number of colonies developed after exposure to PVF was compared to the untreated control.

Cell morphology analysis

HCT116 and L929 cells were seeded in 35 mm Petri dishes (2 × 10⁵ cells/well), treated with 5 μg/mL and 10 μg/mL PVF for 48 h and the morphological changes in response to PVF were observed under a phase contrast microscope (Carton 100 FS) and imaged.

Flow cytometry for cell cycle analysis

HCT116 cells treated with different concentrations of PVF (2.5, 5.0, and 10 μg/mL) and cur (9 μg/mL) as positive control, for different time intervals (24 h and 48 h) were harvested, washed with 1X phosphate buffered saline (PBS), and permeabilized with 70% ice-cold ethanol for 30 min. The permeabilized cells were then treated with 100 mg/mL RNAase A and 50 mg/mL propidium iodide (PI) and subjected to flow cytometric analysis [fluorescence-activated cell sorting (FACS) Aria™, BD Bioscience].

Annexin V-PI staining

Apoptotic cells in response to PVF treatment were visualized with the help of fluorescent microscopy by Annexin V apoptosis detection kit following the manufacturer's instructions (BD Pharmingen 556547, BD Biosciences, USA). Briefly, the HCT116 cells were seeded in a 96-well plate and treated with different concentrations of PVF (2.5 μg/mL and 5.0 μg/mL), and cur (9 μg/mL) as positive control for 24 h. The cells were washed with 1X PBS and 1X assay buffer and incubated for 15 min with 5 μL of Annexin V fluorescein isothiocyanate (FITC) and 10 μL of PI. At the end of incubation, following a PBS wash, fluorescent images were captured using an inverted fluorescence microscope (Nikon TE-Eclipse 300).

Fluorescent microscopy to detect reactive oxygen species

Reactive oxygen species (ROS) levels within the HCT116 cells in response to PVF treatment were observed by 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) staining as per the manufacturer's protocol. Briefly, the cells were seeded in 60 mm dishes and treated with different concentrations of PVF for 6 h. Post-treatment, the cells were harvested and incubated in dichlorodihydrofluorescein diacetate (DCFDA) containing assay buffer for 30 min. The fluorescence developed was imaged and quantified using an inverted fluorescence microscope (Nikon TE-Eclipse 300). Tert-butyl hydroperoxide (TBHP) 50 μmol/L was used as a positive control.

Immunoblot analysis

Briefly, 5 × 10⁵ cells were seeded and treated with different concentrations of PVF (2.5, 5.0, and 7.5 μg/mL), and cur (9 μg/mL) for 24 h. The whole-cell lysates prepared (60–80 μg) were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride (PVDF: Merck IPVH00010) membranes. Each membrane was blocked with 5% skimmed milk for 60 min and probed with labeled primary antibodies against caspase 8, caspase 9, caspase 3, PARP, BID, AIF, p53, BAX, and phospho-AKT. The bands were visualized by staining with 3, 3′-diaminobenzidine or enhanced chemiluminescence reagent [19].

Statistical analysis

The data analysis of flow cytometry was performed through BD FACS Diva software version 5.0.2. All the other statistical analysis was carried out using GraphPad Prism (version 8.0, San Diego, CA, USA) and the quantification of the immunoblot data was performed using ImageJ software (version 1.53 K). Statistical significance was defined as P < 0.05. The error bars represent standard deviation (SD), taken from three independent experiments.

PVF, a bioactive fraction isolated from P. vettiveroides leaves is highly efficacious against colorectal cancer cells

To explore the anticancer potential of the plant P. vettiveroides, we conducted serial exhaustive extraction of the shade-dried leaf powder of the plant, using three organic solvents based on increasing polarity (n-hexane, ethyl acetate, and methanol). All three extracts obtained were tested for the cytotoxic potential in cancer cell lines of various tissue origins using an MTT assay (Figure 1A–C). The results revealed ethyl acetate extract as the most potent, exhibiting notable cytotoxicity with an IC₅₀ value of about 25 μg/mL specifically in the carcinoma cell lines, HCT116 (colon) and HeLa (cervical) (Figure 1D). To isolate the anticancer principle(s) from the ethyl acetate extract, we performed silica gel column chromatography, tested the isolated fractions for cytotoxic activity, and identified a potent cytotoxic fraction, which was designated as “PVF” (Figure S1 and Figure S2, systematic PVF isolation has been detailed in the methodology). Further, we investigated the cytotoxic efficacy of the bioactive fraction, PVF against various human cancer cell lines viz. A375 (melanoma), HCT116 (colon), HeLa (cervical), and PANC-1 (pancreas) using MTT assay. Interestingly, in line with the cytotoxicity profile obtained for ethyl acetate extract, PVF bioactive fraction induced significant cytotoxicity in the colorectal cancer cell line, HCT116, and the cervical cancer cell line, HeLa (IC₅₀—5 μg/mL) (Figure 1E). As P. vettiveroides is used in traditional medicines for gut-related ailments, we continued our studies using colon cancer cell lines. Firstly, we confirmed the increased sensitivity of colon cancer cells towards PVF by screening different colorectal cancer cell lines of two molecular subgroups, viz. cell lines with CIN phenotype (HT-29 and Caco-2) and MSI phenotype (HCT116). Interestingly, all the colorectal cancer cell lines studied exhibited considerable sensitivity toward PVF. The cell line, HCT116 was repeatedly observed as the most sensitive to PVF and was considered for further studies (Figure 1F). We also confirmed the potential of PVF to inhibit the proliferative propensity of HCT116 cells using the clonogenic assay, keeping cur (9 μg/mL) as a positive control. As expected, the number and size of colonies produced were significantly reduced in the PVF-treated plates, as compared to that of the untreated control, confirming the efficacy of PVF against colon cancer (Figure 1G).

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Figure 1. 

PVF, a bioactive fraction from P. vettiveroides is highly efficacious against colon cancer cell lines. (A–C) Screening for the cytotoxic potential of n-hexane, ethyl acetate, and methanol leaf extracts in cancer cell lines of various tissue origins. Various concentrations of leaf extracts (1, 25, 50 and 75 μg/mL) were used for the experiment; (D) determination of IC₅₀ value of ethyl acetate leaf extract in cancer cell lines of various tissue origins. Various concentrations of ethyl acetate leaf extracts (1, 5, 10, 15, 20, 25, 30 and 50 μg/mL) were used for the experiment; (E) determination of IC₅₀ value of the bioactive fraction obtained from the ethyl acetate leaf extract (PVF) in cancer cell lines of various tissue origins; (F) colon cancer cell lines. Various concentrations of PVF (1, 2.5, 5, 7.5, 10, 15, 20 and 25 μg/mL) were used for the experiments; (G) PVF inhibits the proliferative potential of HCT116 cells as assessed by clonogenic assay. Cur 9 μg/mL was used as a positive control. Data are representative of three independent experiments [mean ± standard error of mean (SEM)] and P-values are calculated using one way ANOVA. ^**** P ≤ 0.0001 and ^*** P ≤ 0.001; no.: relative number of colonies

We further tested the biological safety of PVF, by studying its effect on the normal immortalized cell lines viz. mouse fibroblast L929 and human embryonic kidney HEK-293 cells, using MTT assay. Interestingly, the result revealed that the IC₅₀ concentration of PVF in these cells is three times and four times higher than that in HCT116 cells for L929 and HEK-293 respectively (Figure 2A). Morphological examination of HCT116 and L929 cells treated with 5 μg/mL and 10 μg/mL of PVF for 48 h, clearly showed that L929 normal cells are not affected even after prolonged treatment with a concentration i.e. twice the IC₅₀ in HCT116 cells, confirming the non-toxic nature of the fraction on L929 cells (Figure 2B and 2C). These data demonstrate that the potent bioactive fraction PVF acquired from P. vettiveroides is highly efficacious against colorectal cancer cells, whilst being less toxic to normal cells.

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Figure 2. 

PVF is less toxic to normal cells. (A) Cytotoxicity assessment of PVF in normal immortalized L929 mouse fibroblast cells and HEK-293 human embryonic kidney cells; (B) morphological changes in response to PVF in HCT116 cells; (C) L929 cells (20× magnification and scale bar = 50 μm). Data are representative of three independent experiments and the (mean ± SEM) were calculated

PVF induces an apoptotic mode of cell death in colorectal cancer HCT116 cells

We further investigated the mode of cell death induced by PVF in HCT116 cells. Firstly, we analyzed whether PVF induces cell cycle arrest, by flow cytometry analysis. However, PVF did not influence any stages of the cell cycle, even after prolonged treatment for 48 h at IC₅₀ 5 μg/mL and 10 μg/mL (twice the IC₅₀ concentration) (Figure 3A). Interestingly, there was a considerable rise in the number of cells in the sub-G0 phase, which is an indication of apoptosis. To assess apoptotic induction by PVF in HCT116 cells, we performed Annexin V FITC/PI staining. Indeed, upon treatment with PVF, a substantial increase in the number of apoptotic cells stained with FITC/PI+ was noticed compared to that of the untreated control. Cur (9 μg/mL) was used as the positive control (Figure 3B). Many chemotherapeutic drugs currently used in the clinic, invoke apoptosis in cancer cells via ROS-mediated deoxyribo nucleic acid (DNA) damage and p53 activation [20]. Therefore, we tested whether PVF induces ROS generation in HCT116, for which we used ROS-sensitive H2DCF-DA fluorescence assay. Interestingly, PVF treatment in HCT116 cells triggered ROS production as evidenced by the oxidation of H2DCF-DA to dichlorofluorescein (DCF) which led to the generation of green fluorescence, the intensity of which was measured by confocal microscopy (Figure 3C). Together, these results indicated that the cytotoxic mechanism exhibited by PVF involves ROS production, subsequent DNA damage, and apoptosis.

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Figure 3. 

Analysis of the PVF-induced cytotoxic mechanism in HCT116 cells. (A) PVF does not affect any phases of the cell cycle in HCT116 cells as demonstrated by flow cytometry; (B) the extent of apoptosis induced by PVF in HCT116 cells by Annexin V/PI staining (10× magnification and scale bar = 50 μm). Cur 9 μg/mL was used as a positive control; (C) ROS production in response to PVF treatment in HCT116 cells as detected by fluorescence microscopy (20× magnification and scale bar = 50 μm). TBHP 50 μmol/L was used as a positive control. Data are representative of three independent experiments (mean ± SEM) and P-values are calculated using one-way ANOVA. ^**** P ≤ 0.0001, ^*** P ≤ 0.001, and ^** P ≤ 0.01

PVF induces caspase-dependent apoptosis involving intrinsic and extrinsic pathways in HCT116 cells

To further confirm apoptosis and delineate apoptotic pathways in response to PVF in HCT116 cells, we examined the activation of caspases by western immunoblot analysis. Apoptosis in cells is mediated by caspases belonging to the cysteine-aspartic proteases family, classified as initiators and executioners based on their function. The initiator caspases, caspase 8 and caspase 9, activated in response to extrinsic and intrinsic apoptotic stimuli respectively, lead to subsequent activation of executioner caspases, resulting in the cleavage of the cellular death substrates, causing the cells to undergo apoptosis. Upon treatment with PVF in HCT116 cells, we observed both the initiator caspases 8 as well as 9 activations in a dose-dependent manner (Figure 4A and 4B).

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Figure 4. 

PVF induces apoptosis involving intrinsic and extrinsic pathways in HCT116 cells. (A–F) PVF potentiates the activation of caspases 8, 9, cleavage of BID, AIF release, cleavage of caspase 3 and PARP in HCT116 cells as analyzed by immunoblotting; (G, H) PVF induces p53 and BAX activation in HCT116 cells; (I) PVF inhibits phospho-AKT (p-AKT) in HCT116 cells. Cur 9 μg/mL was used as a positive control. Data are representative of three independent experiments (mean ± SEM) and P-values are calculated using one-way ANOVA. ^**** P ≤ 0.0001, ^** P ≤ 0.01, ^* P ≤ 0.1 and ns: P ≥ 0.05

Additionally, we looked for BID activation, a particular proximal substrate of caspase 8 in the extrinsic apoptotic signaling cascade, and a regulator of caspase 8-induced mitochondrial stimulation. Remarkably, we noticed activation of BID upon PVF treatment (Figure 4C), demonstrating amplification of apoptotic signals generated at the death receptor through the mitochondria-mediated intrinsic pathway of apoptosis. Indeed, we observed increased expression of AIF in response to PVF in a dose-dependent manner, demonstrating mitochondrial membrane damage and release of AIF (Figure 4D). Furthermore, PVF induced cleavage of the executioner caspase, caspase 3, and its classical substrate, PARP (Figure 4E and 4F). Conclusively, these data demonstrate that the bioactive fraction (PVF) from P. vettiveroides, induces caspase-dependent apoptosis involving both the extrinsic and intrinsic pathways, in HCT116 cells.

Our next attempt was to investigate the molecular mechanism of the anticancer activity of PVF in HCT116 colon cancer cells. Colon cancer cells of MSI phenotype are reported to show resistance to DNA-damaging agents [21]. Proteins in the DNA mismatch repair (MMR) system are believed to be essential for DNA damage-induced p53 activation and subsequent cell cycle arrest or apoptosis [22]. As HCT116 is an MSI, with wild-type p53, we looked for p53 activation in response to PVF by western immunoblot analysis. Curiously, we observed increased p53 levels in response to PVF in a dose-dependent manner in HCT116, despite being MMR mutant (Figure 4G). We further looked for the transcriptional activity of p53 by analyzing the expression levels of the pro-apoptotic protein BAX, the transcription of which is regulated by p53 [23]. The result of immunoblot analysis showed increased expression levels of BAX protein in response to PVF in a dose-dependent manner, confirming the increased transcriptional activity of p53 (Figure 4H). The data demonstrate DNA damage-induced p53 signaling in response to PVF-induced ROS generation, in HCT116 cells, despite being MMR mutant. Thus, it can be concluded that strong pro-apoptotic signals initiated extrinsically as well as intrinsically in response to PVF in HCT116 drive the cells toward apoptosis. Further, we investigated whether PVF is regulating the anti-apoptotic PI3K signaling in HCT116 by immunoblot analysis of p-AKT. p-AKT inhibits apoptosis by phosphorylation inactivation of pro-apoptotic BAX and BCL-2 associated agonist of cell death (BAD) [24]. It also inactivates the forkhead box O (FOXO) transcription factor, thus down-regulating the expression of BCL-2 interacting mediator of cell death (BIM) and Fas cell surface death receptor (FAS) ligands [25]. Interestingly, we found a significantly high level of p-AKT in HCT116 cells, indicating strong anti-apoptotic signaling, which could make the cells resistant to chemotherapy [26]. However, PVF treatment almost completely ablated the p-AKT band, in HCT116 cells, as assessed by immunoblot analysis (Figure 4I). To conclude, the data clearly shows the ability of PVF in promoting apoptosis in HCT116, by inducing strong proapoptotic signals and inhibiting anti-apoptotic signaling.