Tinospora sinensis (Lour.) Merr alkaloid rich extract induces colon cancer cell death via ROS mediated, mTOR dependent apoptosis pathway: “an in-vitro study”

HCT-116, MCF7, and Hela cells obtained from the National Center for cell science (NCCS, Pune), Dulbecco’s modified eagle medium (DMEM- High Glucose), fetal bovine serum (FBS), MTT reagent (5 mg/ml), phosphate-buffered saline (PBS) procured from Hi-Media. India. Camptothecin, dimethyl sulfoxide (DMSO) supplied by Sigma-Aldrich. Propidium iodide, acridine orange, 2,7-dichlorofluorescein (DCFDA acquired from Thermo Fischer, USA. Primary antibodies like caspases (3, 7, 9), phospo Akt, Akt, phospho ERK, ERK, mTOR, Bcl-2, β-actin, and anti-mouse secondary antibodies were acquired from cell signaling technology (CST).

The collected root material was dried under shade and made into fine powder. Weighed 500 g of the powdered plant material and was subjected to successive solvent extraction using different polarity solvents such as n-hexane, ethyl acetate, and ethanol by the maceration method. Subsequently, each extracted content was filtered using Whatman No. 1 filter paper and evaporated using a rotary vacuum evaporator, and finally, all the extracts were stored at 4 °C till further use. The same procedure was replicated three times for each extract. In the end, the respective extract was combined and stored properly until usage. One hundred milligrams of each solvent extract was accurately weighed and dissolved in 10 ml of DMSO (dimethyl sulphoxide). These DMSO stocks were stored at − 20 °C until use. In all the cell experiments, the final concentration of DMSO was maintained at less than 0.25% (a non-toxic concentration).

After extraction, about 1 mg of each extract of n-hexane, ethyl acetate, and ethanol solvents, was taken for analyzed the presence of various primary and secondary metabolites like carbohydrates, proteins, glycosides, alkaloids, flavonoids, terpenoids, tannins, saponins and polyphenols using various standard methods. The obtained results were tabulated and shown in Table 1.

The n-hexane extract was subjected to GC-MS analysis (GC-MS SHIMADZU GC-2010) using a short (< 5 m) sin column with a constant mass flow rate of carrier gas (helium). Initially, the oven temperature was maintained at 70 °C for 2.0 minutes, and the temperature was gradually increased up to 300 °C at 10.0/35.0 minutes and 4.0 μl of the sample was injected for analysis. The flow rate of helium gas was set to 1 .5 ml/min. The sample injector temperature was maintained at 260 °C and the split ratio was 20 throughout the experiment. The ionization was done with 70 eV. Finally, the mass spectra were recorded for the mass range of 40–1000 m/z for about 35 minutes. The chromatogram and mass-to-charge ratio spectra were used in the identification of compounds [28].

The day before the experiment, all the cancer cells such as HeLa, HCT-116, and MCF-7 cells were plated in the 96-well plate and allowed to grow to reach 70% confluence. Subsequently, the cells were treated with DMSO (vehicle control), with different concentrations of each extract (n-hexane, ethyl acetate, and ethanol) like 50, 150, 250, 350, and 450 μg/ml. Then incubate each extract-treated cell for about 24 h. After the stipulated time, the MTT reagent of 0.5 mg/ml concentration was added to each well and kept for 3 h. After 3 h MTT reagent was removed and added 300ul of DMSO and kept under continuous shaking for 20 min. Finally, the absorbance value was obtained using an ELISA reader at 570 nm. Afterward, the % inhibition for each concentration for each extract against test cell lines was obtained using Graph pad prism version 5.0 and acquired the IC50 value of each extract on each cancer cell [10].

Based on the MTT assay result, the HCT-116 cells were seeded in 12 well plates at a density of 1 × l0⁵ cells/well and subjected to starvation for 6 hours before treatment. After a starvation period, the cells were treated with DMSO control and n-hexane extract (at IC-50 concentration) for 24 hours. Then cells were collected, washed with PBS, and fixed in 70% ethanol at − 20 °C. Fixed cells were stained with PI buffer of about 350 μL at 37 °C for 30 min in a dark place, and then finally analyzed using FACS [29].

HCT-116 cells were seeded in 24 well plates at a density of 5 × 10⁴ cells/well and incubated for 24 hours with vehicle control (DMSO) and n-hexane extract at IC-50 concentration. After 24 hours, added the acridine orange-ethidium bromide of 100 μg/ml was to each incubated cell to distinguish the live, apoptotic, and necrotic cells in treated and untreated cells using a fluorescence microscope with excitation (488 nm) and emission (550 nm) at 200X magnification. Finally, results were obtained and plotted [30].

HCT-116 cells were grown in a 24-well plate at a seeding density of 5 × 10⁴ cells/well and treated with DMSO control, n-hexane extract (at IC-50 concentration), and a standard (Camptothecin 15 μM). After 24 hours of incubation time, the media was removed and washed with PBS twice. A volume of 200 μL of 0.2% triton X was added and incubated for 10 minutes. Excess, triton X was removed and stained with 1 μM of DAPI and observed under the fluorescence microscope with an excitation wavelength of 359 nm and emission wavelength at 460 nm using a DAPI filter at 200 X magnification. The results were obtained and plotted [31].

HCT-116 cells were plated in a 6-well plate at a density of 3 × 10⁵ cells and treated the cells with DMSO, n-hexane extract (at IC-50 concentration), and standard compound (Camptothecin 15 μM) for about 24 h. At the end of the treatment, the medium was removed and washed with PBS. Afterward, the cells were trypsinized with a trypsin-EDTA solution, and then cells were collected in fresh separate 1.5 ml centrifuge tubes. To these cell pellets, 1 ml of chilled 70% ethanol was added and fixed the cells for about 30 min on the ice. To the ethanol-fixed cells, recommended concentration of FITC caspase 3 antibodies was added and incubated for about 30 min at room temperature in a dark place. After the incubation time, the cells were washed with PBS, 0.1% sodium azide was added, mixed thoroughly, and analyzed using FACS [32].

HCT-116 cells were plated in a 12 well-plate and allowed to grow to reach 70% confluence and were treated with DMSO control and n-hexane extract (50 μg/ml) and a standard compound (15 μM). After a specific incubation time, a JC1 stain of 3 μM concentration was added to the wells and incubated for 45 minutes. Then, the cells were trypsinized and centrifuged for 5 min at 1610×g. The pellet was collected and suspended in PBS. Finally, the data was acquired using FACS and plotted [33].

The day before the experiment, HCT-116 cells were seeded in a 12-well plate at a density of 1.5 × 10⁵/well. The next day, the media was changed and treated with vehicle control (DMSO), n-hexane extract (50 μg/ml), and standard camptothecin (15 μM) for 24 h. After the incubation time, the cells were trypsinized and centrifuged at 2516×g for 3 minutes. The cell pellet was collected and washed with PBS buffer. Then, processed for apoptosis with Alexa fluor™ 488 Annexin V/Dead cell apoptosis kit according to the manufacturer’s protocol [33].

HCT-116 cells were seeded uniformly in a 60 mm dish and treated with DMSO, n-hexane extract, and standard compound for 24 h. After the incubation time, the cells were homogenized using RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a complete protease inhibitor cocktail). The lysed samples were centrifuged at 14489×g for 10 min, and the supernatant was collected separately. Finally, the protein was measured by the BCA method and stored at -80 °C till use. The protein sample of the equal amount was loaded and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the immunoblotting was done. The primary antibodies such as caspases (7, 9) mTOR, p-Akt, p-ERK, and Bcl-2 were used at 1:1000 dilutions and the β-actin antibody was used at 1:5000 dilutions. At the end of the experiment, the data was obtained and represented in fold change in comparison with the control as well as standard [10, 34].

The results from all series of experiments were performed in triplicates (n = 3) and expressed as mean ± standard error mean using multiple comparisons of significant analysis of variance (ANOVA) followed by Dunnett’s test as post parametric test using a computer-based fitting program (Prism graph pad version 5.0). A p-value of less than 0.05 is considered statistically significant (compared with the control). Moreover, all methods were carried out according to the relevant guidelines and regulations.

The crude extracts obtained from Tinospora sinensis root were resinous, and the respective percentage yield of n-hexane (NHTR), ethyl acetate (EATR), and ethanol (ELTR) extracts are 6.2, 8.9 and 8.4%. After extraction, each extract was analyzed for the presence of various classes of phytoconstituents. The preliminary phytochemical analysis data revealed the presence of various phytoconstituents in different extracts as shown in Table 1. From the data, it was inferred that an abundant amount of alkaloids was present, more specifically the NHTR enriched with higher alkaloidal contents when compared to other extracts. The further identification of alkaloids in the NHTR was performed using analytical experiments such as GCMS and FT-IR analyses.

Each of the extracts was treated with cancer cell lines like HCT-116, MCF-7, and Hela cells at different concentrations such as 0, 50, 150, 250, 350, and 450 μg/ml. DMSO was used as a control. The MTT assay was carried out to identify the % inhibition on the growth of cancer cells. The MTT assay result revealed the NHTR extract as the most active in comparison with the other extracts (EATR and ELTR) (Fig. 1). The IC-50 values (Shown in Table 2) disclosed that NHTR was more specifically targeting the colon cancer cells (HCT-116 cells), but was less active on Hela and MCF-7 cells (Fig. 1). The IC-50 values of NHTR on HCT-116, Hela, and MCF-7 cells were 42.8 ± 680, 208.0 ± 16.64, and 541.2 ± 2.005, respectively. This assay revealed that the abundant phytochemicals present in the NHRT plays role in producing a cytotoxic effect in HCT-116 cells. Hence, further, the following experiments were carried out on HCT-116 cells at IC-50 concentration.

Several reports stated that plant constituents induce apoptotic cell death in cancer cells either directly or by triggering cell cycle arrest [29, 35, 36]. From the MTT assay, we identified the NHTR as a potent extract for its cytotoxicity, further it also induces cancer-specific cell death in the HCT-116 cells. To identify the mechanism of NHTR in HCT-116 cells, a cell cycle analysis experiment was conducted. FACS analysis was done using control (DMSO), NHRT (50 μg/ml), and standard (Camptothecin;15 μM). The results revealed that NHRT induces significant cell death in HCT-116 cells via arresting the cell division at G0/G1 phase (Fig. 2). Moreover, there was a significant increase in the cell population at G0/G1 phase in NHRT treated group as compared with the camptothecin (15 μM) treated sample. This indicated that NHRT induces cell cycle arrest in HCT-116 cells.

The previous experiment results concluded that the NHRT triggers cell death by inducing cell cycle arrest at G0/G1 phase. However, the mechanism of cell arrest was not explored. Therefore, AO/EB double staining experiment in HCT-116 cells was performed for NHRT using control, and standard. The results indicate that there was a significant decrease in the number of cells in the NHTR-treated group and camptothecin 15 μM (standard) group as compared to the control (DMSO) group. In addition, the images show distinct apoptotic features such as plasma membrane blebbing (late stage of apoptosis), condensed nuclei, and apoptotic body formation. Similar to the standard, the NHTR-treated group showed highly intense red color stained cells significantly in comparison to the control group (Fig. 3). This concluded that the extract induces cell death by apoptosis mechanism. Hence, for further confirmation, we also performed the DAPI staining of the cells. The results showed a notable change in the nucleus of the NHTR-treated groups similar to the standard group. The observed changes are membrane blebbing, formation of horseshoe shape nuclei, and fragmentation. This confirmed that NHRT triggers apoptosis in HCT-116 cells (Fig. 4). In the same way, Annexin V/PI staining of HCT-116 cells was performed for NHRT and standard groups. The results indicated a remarkable increase in the percentage of apoptotic (early and late) cells in the NHRT extract group (28.87%) and standard group (41.76%) as compared with the control group (10.06%) (Fig. 5).

Besides, NHRT also induces caspase-3 expression in the HCT-116 cells as compared with the control (Fig. 6). Generally, the M1 quadrant represents many living cells with no activation of caspase-3, whereas the M2 quadrant represents apoptotic cells with activated caspase-3. In the experimental result, there was a significant increase in the cell population in the M2 quadrant specifically in NHRT and standard-treated cells. On the other hand, there was no increase in cell population in the M2 quadrant in the control group (Fig. 6). This concluded that NHRT induces apoptosis in HCT-116 cells via caspase 3 activation. In continuation, we experimented to establish the relation of caspase 3 activations to mitochondrial dysfunction in apoptosis. Here, we found that NHRT and standard groups significantly reduce the mitochondrial membrane potential (MMP) as compared to the control group, where JC-1 acts as an indicator of MMP (Fig. 7). In this experiment, the NHRT suppressed the MMP, thus indicating the accumulation of the JC-1 monolayer (Blue fluorescence). These results confirmed that NHRT triggers apoptosis in HCT-116 cells through mitochondrial dysfunction. It was evident in the DCFDA experiment where NHRT induced upregulation of intracellular ROS and mitochondrial dysfunction in HCT-116 cells (Fig. 8).

In colon cancer cells, the mammalian target of rapamycin (mTOR) is always in an active state and is mainly responsible for cell growth, survival, and metabolism. Moreover, mTOR is the major downstream target of the PI3K/AKT pathway. In a stress environment, the cell incorporates both the caspase family and mTOR signaling for regulating cellular function [37]. At this juncture, inhibition of mTOR and its associated signals along with activation of caspases such as 3, 7, and 9 shall lead to cell death. Earlier reports suggested that forced inhibition of mTOR by chemotherapeutics always results in the triggering of apoptosis via activation of caspase-3 function [37]. In our western blot experiments, we found that NHRT (50 μg/ml) and standard (camptothecin 15 μM) activate apoptotic cell death in HCT-116 cells via an extrinsic pathway. The results (Fig. 9 & Fig. S1) revealed that NHRT down-regulates phosphatidylinositol 3-kinase PI3K/Akt signals, and its downstream signal mTOR. The NHRT inhibited the anti-apoptotic protein Bcl-2, furthermore, it up-regulated both caspase 7 and caspase 9 significantly in comparison to the control group. From the literature, it is known that the Ras/PI3/Akt/mTOR pathway is coordinated with the Ras/Raf/Mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway for controlling cell proliferation. Hence, modulation of p-ERK/ERK along with the mTOR and its associated signals leads to apoptotic cell death in cancer cells. Similarly, our experiment results (Fig. 9 & Fig. S1) showed that NHRT inhibited the mTOR pathway in addition to the inhibition of p-ERK, and induced apoptosis [37].

It is known that medicinal plant shows multiple biological functions due to presence of various characteristic phytochemicals. In our study the n-hexane extract also demonstrated a potential anticancer effect against colon cancer cells (HCT-116) specifically. Here, it is important to identify the actual compounds responsible for the observed anticancer activity. Hence, GC-MS analysis was conducted for NHRT. The results revealed the presence of several compounds in NHRT. A careful examination of peaks in the chromatogram, and their m/z value confirmed the presence of a few alkaloids and other compounds, namely tembetarine (m/z 344.4), berberine (m/z 336.4), magnoflorine (m/z 342.41), palmatine (m/z 352.4), Beta-sitosterol (m/z:414.7), cordifoliside E (m/z:372) and jatrorrhizine (m/z:338) (GC-MS chromatogram and retention time of respective compounds (Shown in Fig. 10a&b).

The sample was subjected to FT-IR analysis. The obtained FT-IR spectra (Fig. 11) revealed several regions correspond to characteristic IR absorption (cm^− 1) of alkaloids such as N-H (3423), C-H (3184), C = C, C=O (1600, 1697), C = N (1620), C-N (1335), C-O-C (1056).