According to the aforementioned study, the growth of HeLa cells was significantly inhibited in a concentration- and time-dependent manner on treatment with Rd. The IC₅₀ value of Rd against the HeLa cell line was determined as 150.5 ± 0.8 µg/ml after 48 h of incubation. Also, the cells treated with Rd showed typical apoptotic features characterized by the appearance of apoptotic bodies, volume reduction, and nuclear fragmentation with dense Kelly fluorescence and chromatin condensation when monitored under a fluorescence microscope (Yang et al., 2006).

After the treatment of HeLa cells for 48 h with the ginsenoside Rd, significant changes were observed in the cell cycle distribution at different concentrations (i.e. 120, 180, and 210 µg/ml). The drug-treated cells were characterized by an increase in the S phase cells and a decrease in the G₀/G₁ phase cells in a dosage-dependent manner. However, no obvious changes were detected concerning G₂/M phase cells. After a 48 h treatment of the HeLa cells with Rd, a hypo-diploid (apoptotic) peak of DNA, characteristic of apoptosis, was observed. The corresponding apoptosis rates were determined as 7.4, 15.9, and 35.8% at 120, 180, and 210 µg/ml of Rd, respectively. In the control group, only a minor cell population (ca. 0.9%) was found to undergo apoptosis (Yang et al., 2006).

The function of proteins like Bax and Bcl-2 is to either enhance or inhibit apoptosis. To assess the role of the Bcl-2 family genes in apoptosis induced by the ginsenoside Rd, the expressions of these two proteins were immunohistochemically evaluated in HeLa cells with the aid of the streptavidin ± biotin complex (SABC). It was found that in comparison with the control cells, the Bcl-2 expression was markedly decreased, whereas the Bax expression was enhanced, suggesting that Rd is capable of downregulating the Bcl-2 protein levels while upregulating the Bax protein expression (Yang et al., 2006).

Previous studies have reported that the collapse of mitochondrial transmembrane potential is considered a critical event among all cell types undergoing apoptosis, regardless of the inductive signal. To assess the effect of Rd on the mitochondrial transmembrane, both the control- and drug-treated HeLa cells were stained with Rhodamine 123. It was evident that the mitochondrial transmembrane potential of HeLa cells treated with Rd at concentrations of 180 or 210 μg/ml for 48 h was decreased significantly, indicating that Rd is capable of decreasing membrane potential without altering plasma membrane permeability (Yang et al., 2006).

Caspase-3, in particular, has been reported to have major involvement in the execution of apoptosis and is characterized as the best effector caspase present downstream of Bax and Bcl-2. It is known that the activation of caspase-3 results in cellular death via the proteolytic degradation of various cellular components. To test the viability of cells treated with Rd, the HeLa cells were first incubated with the cell-permeable peptide NH2-Asp-GluVal-Asp-CHO (DEVD-CHO; 2 µM), a highly specific potent inhibitor of caspase-3 and with RPMI-1640 medium (control) for 4-h (Yang et al., 2006). Then, the cells were exposed for 48 h at different concentrations of Rd, and the viability of cells was assessed with the aid of the MTT assay. It was evident that DEVD-CHO significantly increased the viability of the HeLa cell treated with Rd (tested at doses of up to 240 µg/ml) (Yang et al., 2006). Hence, hindering the inhibition of proliferation induced by Rd declares a prominent role of caspase-3 in apoptosis.

The MCF-7 cells were treated with increasing amounts of G-Rh2 to observe dose-dependent inhibition and were scored by using a hemocytometer. An inhibition by 40% and 47% was observed on incubating G-Rh2 with cell line concentration sof 50µM and 100 µM for 48 h. To assess whether perturbing the cell cycle–related events are accountable for such kind of suppressive effects, flow cytometry was applied after staining with propidium iodide (PI) to measure the DNA content and the cell cycle distribution of G-Rh2–treated and G-Rh2–untreated cells. The results presented no effects in the control cells treated with the vehicle but led to a marked accumulation of cells in the G_l phase in MCF-7 cells treated with Rh2. This suggests that growth inhibition was the result of a block during the G_l phase and that such cells did not enter the S phase.

Oh et al. (1999) observed that G-Rh2 inhibited the proliferation of MCF-7 in a dose-dependent manner. This effect was reversible. Lee et al. (2018) reported that Rh2 causes methylation in genes responsible for immune response and tumorigenesis. This phenomenon not only leads to greater immunogenicity but also inhibits cell growth. Rg3 also caused the apoptosis of MCF-7 cells via the inhibition of NF-κB (Kim et al., 2014).

The expression of cell cycle–regulating proteins at the G_l/S junction, such as D-type cyclins, cyclin E, Cdk2, Cdk4, and p21 were determined to further assess the mechanism by which G-Rh2 arrests cells in the G_l/S transition phase via Western immune blot (Oh et al., 1999). The results indicated constant expression levels of Cdk2, Cdk4, and cyclin E; however, a decrement in the level of cyclin D3 (10 µM concentration) by G-Rh2 was observed in a time-dependent manner. Next, G-Rh2 was examined as a potential inducer of Cdk inhibitors. A marked increase in the expression of the Cdk inhibitor p21 protein at a dosage higher than 10 µM was observed after Rh2 treatment. Similarly, significant increment in the binding of p21 and Cdk2 was also very prominent. To our advantage, no effect over the expression levels of tumor-suppressive protein p53 and p27, which are the members of the CIP/KIP family, was displayed.

As discussed earlier, Cdk2 kinase activity (activated by binding with cyclin E) is essential for G_l–S phase progression. As the G_l/S phase of the cell cycle was perturbed by Rh2, there was no effect over the expression levels of Cdk2 and cyclin E. Hence, to verify that whether G-Rh2 inhibits the Cdk2 and cyclin E–dependent kinase activities, histone H1 was used as a substrate to assess the Cdk2 kinase activity which was found to be significantly inhibited in response to G-Rh2 treatment. At 12 and 24 h of G-Rh2 treatment, 20% and 80% decrease was observed in Cdk2 kinase activity, respectively. Similarly, 80% reduction in the activity of cyclin E–dependent kinase was noted after treatment with G-Rh2 following the time interval of 24 h as compared to the untreated control (Choi et al., 2009). These results demarcate that the suppressive effect of G-Rh2 on the cell growth of MCF-7 cells has been partially due to downregulating the activities of Cdk2 and cyclin E–dependent kinase rather than altering the expression of their proteins.

The genetic makeup of every cell tends to play a vital role in cell cycle phase transitions. The product of the Rb gene known as pRb is an important frontier protein in the G_l phase of the cell cycle. A tentative relationship was hypothesized as “whether G-Rh2 link decreased the expression of D-type cyclins is associated with the kinetics between the phosphorylation of the pRb and E2F family of MCF-7 cells.”

Molecular analysis has revealed a remarkable decrease in the pRb levels of the expression and was transformed from the hyper-phosphorylated form at 0 h to hypo-phosphorylated or un-phosphorylated form by G-Rh2. Similarly, a time-dependent decrease in the level of phosphorylation of p130 was observed associated with G-Rh2 treatment. Simultaneously, an immunoprecipitation assay has revealed a strong increase in the association of pRb with the transcription factor E2F-1 and enhanced the binding of p130 and E2F-4 after G-Rh2 treatment whereas undetectable association between pRb and E2F-1 was reported among the untreated control cells, indicating that the release of the E2F family gets inhibited after Rh-2 treatment (Table 1; Oh et al., 1999).

To assess the growth inhibition characteristics of Rh4, the mice were inoculated with Caco-2 cells so that a colorectal xenograft model can be established. The mice treated with 40 mg/kg of CAMPTO (Irinotecan) and 20–40 mg/kg of Rh4 exhibited small tumors than the control group after 30 days of treatment. The body weight, however, in both the cases showed an appreciable difference presenting normal weight in Rh4-treated cells as of lower weight of mice treated with CAMPTO, suggesting that Rh4 exhibits little cytotoxicity and fewer side effects (Wu Q. et al., 2018).

The hematoxylin and eosin staining of tumor tissues and major organs presented a significant decrease in tumor cells with no obvious organ toxicity. Similarly, a histological analysis revealed no obvious injury such as enlargement of the glomerulus, degenerative changes to the cardiac structure, disordered architecture of the hepatic lobule, or collapse of the alveoli. Furthermore, the CCK-8 assay established colorectal carcinoma cells such as Caco-2 and HCT-116 which were significantly inhibited by Rh4 in a concentration- and time-dependent manner (Wu Q. et al., 2018).

To analyze the functional role of Rh4 with its anti-colorectal cancer effect, a flow cytometry analysis was conducted. The analysis revealed that after incubation for 24 h with 240 µM, the cells were found to increase in the G₀/G₁ phase by 39.06% in the Caco-2 cell line and 31.05% in HCT-116 cell line, respectively, whereas reduction in the number of Caco-2 cells by 18.87% was observed in the S phase while no appreciable difference in the S phase of the HCT-116 cell line was noted. The expression of cell cycle proteins including cyclin D and cyclin-dependent kinase 4 (CDK4) were further analyzed via Western blotting, and the results showed significant reduction in their expression levels, whereas the levels of p53 and p21 on the other hand were enhanced in a dose-dependent manner (Wu Q. et al., 2018).

To further assess the apoptotic pathway induced by Rh4 (that is,intrinsic or extrinsic), the apoptosis-related proteins were investigated by Western blotting. It was observed that Rh4 treatment leads to the upregulation of the cytosolic cytochrome C, Bax and caspase- 3 and caspase- 9 levels and downregulation of the mitochondrial cytochrome C, indicating the involvement of the intrinsic pathway (Table 1). Similarly, the levels of cleaved-caspase-8 increased inversely proportional to levels of Fas, confirming the participation of the extrinsic pathway. Hence, both pathways have been found in triggering the mechanism of apoptosis (Wu Q. et al., 2018).

Previous studies have revealed that steamed EPN can induce ROS generation, in addition to cell death (Kachadourian et al., 2010). To assess the production of ROS on treatment with Rh4, the levels of ROS were measured in Caco-2 cells. An elevated level of ROS was recorded in a concentration-dependent manner on conducting the DCFH-DA assay after getting treated Rh4 in the Caco-2 cell line. On a further note, the study elucidated the anti-cancer mechanism of Rh4 on the MAPK-p53 signaling pathway.

A heightened expression of phosphorylated JNK and p53 was observed in a dose-dependent manner after treatment with Rh4, but a very minimal effect was noticed over the phosphorylation of ERK and p38 in both Caco-2 and HCT-116 cell lines. To assess the relationship between ROS and JNK/p53, the expression of p-JNK and p-p53 was followed by pretreatment with NAC, SP600125, and PFT-α. The results showed the attenuation of JNK and p53 phosphorylation by NAC and SP600125 while an inhibition of the p53 expression in Caco-2 cells, indicating that JNK is a downstream target of accumulated ROS, and p53 serves as a downstream effector in response to activated JNK.

CSCs have been known to self-renew and grow as non-adherent spheres, and the cytotoxic effects of Rb1 and its metabolite compound K were examined over SKOV-3 and HEYA-8 cancer stem cells. The formation of small spheres was observed on treatment with Rb1 and compound K in comparison to control cells in a dose-dependent manner. Similarly, trypan blue exclusion assay revealed a 1.5-fold increase in cell death for Rb and compound K in SKOV-3- and 1.4-2-fold increase in the cell death of HEYA-8 cell lines, respectively.

In addition, Rb1 and compound K accounted for progression in the rate of apoptosis by 5.9- and 9.6-fold in SKOV-3, whereas 1.6- and 2.9-fold in HEYA-8 by expressing the active caspase-3, suggesting that apoptosis may lead to loss in cell viability. Subsequently, cancer stem cells were treated with 250 nM Rb1 and 125 nM compound K for different periods (0, 24, and 48 h) following up with the expression of CSC markers. Bmi-1, Nanog, and Oct4 were three identified ovarian markers that were significantly depleted in a time-dependent manner with maximal depletion observed at 48 h of treatment (Deng et al., 2017).

As pertaining to our prior understanding, the sphere-forming capability found in CSCs is an indirect representation of viable stem cells, and the experiment further investigated the effects of Rb1 and compound K in the relapse situation. SKOV-3 and HEYA-8 primary spheroids were chosen along with the control vehicle and treated with 250 nM Rb1 or 125 nM compound K and then were dissociated and replated as secondary spheroids. By day 2, the control group started to develop into secondary spheroids, whereas the cells treated with compound K and Rb1 did not. However, their removal from SKOV-3 and HEYA-8 CSCs did not restore the secondary spheroid formation activity of CSCs representing irreversible damage (Deng et al., 2017).

As of now, other potential drug candidates and compounds of this genus have been known to produce a synergistic effect in combination with other chemotherapeutics. To investigate this effect of Rb1 and compound K on an individual and combinatorial basis, both compounds were used alone or in combination with two frontline drugs in the treatment of cancer (i.e. cisplatin/paclitaxel). It was found that the drugs with relevant doses of 50 and 100 µM had a mild effect of SKOV-3 and HEYA-8 sphere formation. However, Rb1 and compound K in combination with the same doses sensitized the CSCs toward chemotherapeutics (Deng et al., 2017).

It has already been noticed that Rb1 upon oral administration gets metabolized into compound K (Hasegawa et al., 1996). Furthermore, the underlying mechanism of compound K was examined to present the whole process being executed at the back end. Emerging evidence has suggested an intricate role of EMT in CSC self-renewal (Polyak and Weinberg, 2009). To investigate this fact, the levels of EMT transcription factors were determined. The results declared that the two major transcription markers (that is, snail and slug) were significantly downregulated on treatment with compound K in the presence of cisplatin and paclitaxel.

Similarly, E-cadherin (an epithelial marker and tumor suppressor) expression was reverted back to its normal state. Hence, the underlying molecular mechanism by which compound K has caused inhibition of snail and slug was investigated. As for earlier literature, the PI3K/AKT and ERK1/2 mitogen-activated protein kinase pathways are known to play a critical for the activation of snail and slug (Jorda et al., 2007; Suman et al., 2014). Thus, in order to validate the role of the aforementioned pathways, the phosphorylated (active) forms of AKT and ERK1/2 were assessed. The addition of compound K led to a decrease in the phosphorylation of PI3K/AKT and ERK1/2 which might account for the reduced expression of snail and slug (Table 1). Furthermore, to justify this hypothesis, a constitutively active construct of AKT (E17K) and MEK1 (CA-MEK1) was used. Both snail and slug expressions were still observed after the addition of compound K. Thus, the role of EMT was affirmed in compound K mechanism (Deng et al., 2017).

To this extent, the role that compound K plays in the inhibition of CSC survival has still been ambiguous; to further address this issue, molecular analysis was performed. β-Catenin which has been known to play the forefront role in tumorigenesis or self-renewal is an intriguing characteristic (Fodde and Brabletz, 2007; Chien et al., 2009). Therefore, the degree of expression to which β-catenin was produced was analyzed. A marked decline was noted in β-catenin levels on treatment with compound K. β-catenin has also been found to interact with the T-cell factor (TCF) activating various transcriptional genes, and on treatment with compound K, a decrease in the β-catenin/TCF was quite prominent. Similarly, the expression of ABCG-2 and P-glycoprotein drug transporters that are targets of β-catenin/TCF involved in the efflux of chemotherapeutics was also downregulated in SKOV-3 and HEYA-8 cell lines.

The viability of HCT-116 cells in the experiment after treatment with 75, 150, or 300 µM of NGR1 was noted to be approximately the same as that of the control during 48 h of incubation. However, the change in the cells’ viability was observed on increasing the concentration to 500 µM, where NGR1 caused an appreciable reduction (58 ± 7.26%) as that from the control (100%) in the duration of 48 h (Lee et al., 2017).

NGR1 has been found to possess profound effects in the HCT-116 cell line in a concentration- and time-dependent manner at the wound-healing areas. A reduced wound closure rate (17–21%) was observed on treatment of the HCT-116 cell line with 75, 150, or 300 µM NGR1 for 24 or 48 h in comparison with the control group (24–34%). Hence, significant suppression of HCT-116 cell migration was induced by NGR1 (Lee et al., 2017).

Further evaluation was conducted by checking the effects of NGR1 overexpression levels of MMP-2 and MMP-9. The results presented that NGR1 did not regulate MMP-2 expression in the HCT-116 cell line. However, the levels of MMP-9 on treatment with 150 or 300 µM NGR1 for 24 h were significantly decreased by 35 and 68%, respectively, compared with the control group (100%) (p < 0.01). The aforementioned results indicate that NGR1 regulates migration and intravasation by regulating the MMP-9 expression in HCT-116 cell lines, as presented in Figure 2 (Lee et al., 2017).

The property of adhesion among EA. hy 926 cells co-cultured with HCT-116 cells was estimated to get significantly decreased by 18–20% on treatment with 150 or 300 µM of NGR1 for 24 h. To further explore the mechanism, the expression levels of integrin-1 in HCT-116 cells and ICAM-1 plus E-selectin in the EA. hy926 cells were analyzed. The expression levels of all three markers after treatment of HCT-116 cells with NGR1 for 24 h were significantly decreased (Table 1). The level of decrement in comparison to control cells (that is, 100%) was 10–27% in case of integrin-1 and as for E-selectin, the expression levels were 85 ± 5. Similarly, the expression levels were found to be 78 ± 7 and 78 ± 4 after treatment with 75, 150, or 300 µM NGR1 for 24 h in EA. hy926 cells for integrin-1 and E-selectin, respectively (Lee et al., 2017). The corresponding trend of significant reduction with increasing concentration (86 ± 3, 68 ± 3, and 34 ± 2%) was consistent in the case of ICAM-1 compared with the levels in the control cells (100%) (Lee et al., 2017).

To understand the impact of NGR1 on extravasation, changes in HCT-116 cells’ morphology were monitored using scanning electron microscopy. The lipopolysaccharide (LPS)-treated HCT-116 cells reflected a flattened and collapsed appearance, whereas the HCT-116 cells treated in combination with LPS and 300 µM of NGR1 presented a less flattened shape. The study also pursued to monitor the effect of NGR1 on the TEER of EA. hy926 cells. The obtained transepithelial/transendothelial electric resistance (TEER) values were 113 ± 3, 128 ± 14, and 134 ± 16%, after getting treated with 75, 150, or 300 µM NGR1 respectively, signifying an elevation in comparison with the control group (100%). Thus, increase in the TEER value dictates decrement in cell permeability while inhibition of endothelial cells dictates invasion (Lee et al., 2017).

The study presents the exposure of U87 glioma cells for 3 days with 20(S)-Rg3 accounted for dose-dependent inhibition. A concentration ≥10 µM was responsible for significant suppression of the growth of glioma cells whereas lesser than that had a little significant impact. Next, the involvement of apoptosis was evaluated in 20(S)-Rg3–induced growth arrest. Annexin V staining revealed that sub-lethal doses (10–20 µM) had a low profile of apoptotic cells in comparison with high doses (50–100 µM) of 20(S)-Rg3.

20(S)-Rg3 (20 µM) was found to completely subdue cell proliferation for at least nine days causing only modest levels of cell death. Glioma cells were treated with various sub-lethal concentrations of 20(S)-Rg3, and the expression of senescence-associated β-galactosidase (SA-β-gal) was measured to assess whether the cell growth arrest in response to 20(S)-Rg3 has been caused due to the induction of cellular senescence (Sin et al., 2012).

The results obtained presented 67% of U87 cells after chronic 20(S)-Rg3 treatment at a 20 µM concentration came up with positive staining whereas only ∼5% cells were stained positive in the DMSO control. Positive staining for SA- β-gal had a characteristic flattened and enlarged appearance that was consistent with cellular senescence. Hence, these findings suggest that senescence as well as apoptosis can be induced by 20(S)-Rg3. In addition, an immunoblot analysis presented an enhanced expression of p53 and cyclin-dependent kinase inhibitor (P21CIP) in 20(S)-Rg3–treated cells (Sin et al., 2012).

It is evident from the literature that reactive oxygen species (ROS) are accounted as inducers of senescence. It was evaluated whether the cells treated with 20(S)-Rg3 produces any oxidative stress. ROS levels were assessed via the dichlorofluorescein assay. As it is known that the mitochondrial ROS are majorly responsible for elevated ROS content, therefore, it was also estimated via fluorescence by MitoSOX red as a marker of mitochondrial superoxide. The results showed that the intensity of fluorescence was progressively increased in U87 glioma cells, accounting for the elevated intracellular ROS levels. To verify the results, an ROS scavenger (i.e. N-acetyl-1-cysteine) was added before exposure to 20(S)-Rg3. It was noted that the number of SA-β-gal cells was significantly reduced. These corresponding results strongly suggest the role of ROS in contributing to senescence (Sin et al., 2012).

To examine whether the elevation of ROS has any impact over the PI3K/Akt signaling pathway, the phosphorylated Akt levels of the 20(S)-Rg3–treated U87 cells were measured. U87 cells treated with 20(S)-Rg3 was characterized to have elevated levels of phosphorylated Akt levels at 20 µM concentration. To further assess the role of phospho-Akt in the induction of senescence, siRNA against Akt was employed. The results presented depleted levels of Akt, accounting for decreased SA-β-gal activity compared with the control siRNA cells (Table 1). It has also been reported that ROS generation involves the Akt pathway (Dolado and Nebreda, 2008). Hence, it was investigated that whether elevated ROS levels occur due to the activation of Akt or it is a secondary effect produced by the drug. Transfection with siRNA significantly reduced ROS production by 32%, indicating a vital role of Akt in the regulation of cellular ROS and senescence production (Sin et al., 2012).