GC-MS study showed the presence of various bioactive components in the leaves of Tagetes erecta
The results of GC-MS analysis revealed the presence of various bioactive components in the leaves of Tagetes erecta (Fig. 1 and Supplementary Table 1). The major bioactive compounds detected in the leaves of TE were Hexadecanoic acid (Palmitic acid), 9, 12, 15-octadecatrienoic acid (Linolenic acid), Quinic acid (1,3,4,5-Tetrahydroxy-cyclohexane carboxylic acid), 2,3- dihydrobenzofuran (Coumaran), and β-stigmasterol.
AETE administration reduced the tumor volume in tumor-bearing mice
TV of the developing tumor was measured on every alternative day, and it was analyzed in 10 days intervals (Fig. 2a). In the tumor control group, the TV was elevated exponentially in a time-dependent manner. However, treatment with MMC and AETE showed dose and time-dependent reduction in TV. A maximum reduction in TV was observed on the 50th day when the animals were treated with a high dose of AETE (400 mg/kg BW). By the 50th day, the TV of tumor-bearing animals without any treatment (tumor control group) was 19.74 ± 1.81 cm3. However, MMC treatment significantly reduced the TV to 13.34 ± 1.17 cm3 (p < 0.001 vs. tumor control). Further, AETE treatment up to the 50th day showed the most prominent result. The EAC-induced TV was significantly reduced to 7.83 ± 0.91 (p < 0.001 vs. tumor control) and 3.00 ± 0.28 (p < 0.001 vs. tumor control) when the tumor-bearing animals were treated with 200 mg/kg BW and 400 mg/kg BW of AETE, respectively.
AETE administration led to an increase in survival time of tumor-bearing animals
To assess the effect of AETE on life span, tumor-bearing animals were treated with MMC (2 mg/kg BW) and AETE (200 mg/kg BW and 400 mg/kg BW) for 50 days. The experimental animals were kept under regular supervision, and the death pattern of the animals was recorded. The survival time of tumor-bearing animals was counted from the day of AETE administration. In the present study, we observed that both MMC and AETE treatment had an apparent effect on experimental animals' survival time compared to tumor control animals. All the animals of the tumor control group have expired within 62 days (Fig. 2b). MMC treatment led to an increase in the survival time by 182.44%, with a survival of at least 40% of animals more than 100 days. AETE administration showed a dose-dependent increase in life span, and AETE treatment (200 mg/kg BW) showed an increment in survival time by 328.31%, with at least 30% surviving animals for more than 200 days. AETE administration at high dose (400 mg/kg BW) showed more improvement, and the life span of tumor-bearing animals was increased by 476.86%, and 50% of animals were survived more than 280 days (Fig. 2b).
AETE treatment led to an increase in body weight gain in tumor-bearing animals
The BW of the experimental animals was recorded on every alternative day for entire experimental periods. The effect of AETE administration on the BW of experimental animals was analyzed on the 30th and 50th days of AETE treatment. On the 30th day of AETE exposure, we observed a BW gain of 13.65% in the control group (Table 1; Fig. 2c). However, BW decreased drastically in the tumor control group, and a BW loss of 9.80% from initial weight was observed (p < 0.001vs. control) (Fig. 2c). MMC and AETE treatment had a substantial effect on BW, and the BW loss of tumor-bearing mice was recovered extensively upon treatment. In MMC treated group, the BW gain was elevated to 6.55%. Administration of AETE at a dose of 200 mg/kg BW and 400 mg/kg BW resulted in BW gain in tumor-bearing animals, and it was 9.75% and 10.57%, respectively (Table 1). In the present study, we observed a dose and time-dependent effect of AETE on BW of experimental animals. On the 50th day of AETE treatment, the BW gain in control animals was 22.45% (Table 2). The tumor-bearing animals of the tumor control group had a significant BW loss of 11.83% (p < 0.001 vs. control) (Fig. 2c). Treatment of MMC and AETE showed a considerable improvement in BW gain. We observed a BW gain of 8.96% (p < 0.001 vs. Tumor control) (Table 2; Fig. 2c) in MMC-treated animals. In this study, we noticed that AETE treated animals had gained more BW as compared to MMC treated group. The BW gain in AETE treated animals was 11.07% (p < 0.001 vs. tumor control ) and 21.07% (p < 0.001 vs. tumor control) when tumor-bearing animals were administered with 200 mg/kg BW and 400 mg/kg BW of AETE, respectively.
Table 1
Effect of AETE on body weight and tumor weight in EAC solid tumor-bearing mice at 30th day of AETE administration
Parameters | On 30th day of AETE treatment |
Control | Tumor control | MMC (2 mg/kg) | AETE (200 mg/kg) | AETE (400 mg/kg) |
Initial body wt. (g) | 26.89 ± 1.62 | 26.46 ± 1.05 | 26.35 ± 1.43 | 26.21 ± 0.73 | 27.29 ± 1.85 |
Final body wt. (g) | 30.55 ± 1.66 | 39.47 ± 2.07*** | 36.37 ± 1.27*** | 36.27 ± 1.38*** | 36.41 ± 2.32*** |
Tumor wt. (g) | - | 15.60 ± 1.57a,b,c | 8.30 ± 0.69a | 7.50 ± 0.56b | 6.25 ± 0.71c |
Net body wt. (g) | 30.55 ± 1.66 | 23.87 ± 1.07*** | 28.07 ± 1.40** | 28.77 ± 1.03 | 30.16 ± 1.88 |
Body wt. change (g) | + 3.66 | -2.59 | + 1.72 | + 2.56 | + 2.87 |
% of change in body wt. from initial wt. | + 13.65% | -9.80% | + 6.55% | + 9.75% | + 10.57% |
MMC; Mitomycin C. Values are expressed as mean ± SD of 10 experimental animals (n = 10). Statistical analysis: one-way ANOVA followed by Tukey's test for multiple comparisons. **p < 0.01 vs control; ***p < 0.001 vs control and a,b,cp < 0.001 vs. tumor control group. A positive and negative sign indicates gain and loss of BW, respectively. Body wt. change (g) and % of change in body wt. from initial wt. was calculated by taking the average values. Body wt. change (g) = Net body wt. (g)-Initial body wt. (g). |
Table 2
Effect of AETE on body weight and tumor weight in EAC solid tumor-bearing mice at 50th day of AETE administration
Parameters | On 50th day of AETE treatment |
Control | Tumor control | MMC (2 mg/kg) | AETE (200 mg/kg) | AETE (400 mg/kg) |
Initial body wt. (g) | 27.23 ± 1.83 | 27.08 ± 1.63 | 27.27 ± 1.84 | 27.32 ± 1.58 | 27.30 ± 1.82 |
Final body wt. (g) | 33.33 ± 2.08 | 50.60 ± 1.33*** | 44.24 ± 3.30*** | 41.63 ± 1.53*** | 39.14 ± 2.23*** |
Tumor wt. (g) | - | 28.07 ± 1.25a,b,c | 14.52 ± 1.43a | 11.29 ± 0.88b | 6.10 ± 0.63c |
Net body wt. (g) | 33.33 ± 2.08 | 23.88 ± 1.58*** | 29.72 ± 2.12** | 30.34 ± 1.67 | 33.04 ± 2.03 |
Body wt. change (g) | + 6.10 | -3.20 | + 2.45 | + 3.02 | + 5.74 |
% of change in body wt. from initial wt. | + 22.45% | -11.83% | + 8.96% | + 11.07% | + 21.07% |
MMC; Mitomycin C. Values are expressed as mean ± SD of 10 experimental animals (n = 10). Statistical analysis: one way analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons. **p < 0.01 vs control; ***p < 0.001 vs. control and a,b,cp < 0.001 vs. tumor control group. A positive and negative sign indicates gain and loss of BW, respectively. Body wt. change (g) and % of change in body wt. from initial wt. was calculated by taking the average values. Body wt. change (g) = Net body wt. (g)-Initial body wt. (g). |
Table 3
IC50 value of AETE in EAC and HEp-2 cells at different treatment conditions
| IC50 (mg/ml), 48 h | IC50 (mg/ml), 72 h |
| Trypan blue | MTT | Trypan blue | MTT |
EAC | 0.15 ± 0.03 | 0.20 ± 0.02 | 0.14 ± 0.02 | 0.16 ± 0.02 |
HEp-2 | 0.17 ± 0.03 | 0.47 ± 0.03 | 0.09 ± 0.01 | 0.28 ± 0.02 |
IC50, 50% inhibitory concentration; EAC, Ehrlich ascites carcinoma; HEp-2, Human larynegeal carcinoma |
AETE exposure reduces the tumor weight in solid tumor-bearing animals
The effect of AETE on TW was analyzed on the 30th and 50th days of AETE administration, as mentioned earlier. In the present study, we observed a dose and time-dependent decrease in TW when the animals were treated with AETE. On the 30th day, the TW of MMC treated animals was decreased to 8.30 ± 0.69 g (p < 0.001) as compared to tumor control animals (15.60 ± 1.57 g) (Table 1; Fig. 2d). Upon AETE administration at a dose of 200 mg/kg BW and 400 mg/kg BW, the TW of tumor-bearing animals was 7.50 ± 0.56 g (p < 0.001) and 6.25 ± 0.71 g (p < 0.001), respectively as compared to the tumor control group. Similarly, on the 50th day, the TW of the tumor control group was 28.07 ± 1.25 g, whereas in MMC treated group, it was reduced to 14.52 ± 1.43 g (p < 0.001) (Table 2; Fig. 2d). Significant reduction in TW was also detected when experimental animals were treated with 200 mg/kg BW and 400 mg/kg BW, and it was found to be 11.29 ± 0.88 g (p < 0.001) and 6.10 ± 0.63 g (p < 0.001), respectively (Table 2; Fig. 2d).
The gross morphological appearance of control and tumor-bearing animals at the 30th and 50th day of AETE treatment was shown in Fig. 2e and Fig. 2f. The solid tumor was constructed in the thigh tissues of experimental animals, as shown in Fig. 2 (e-f). Control animals for both the 30th and 50th days showed normal-sized thigh tissues, whereas a vastly over-sized solid tumor was observed in tumor control group animals on both 30th days (Fig. 2e) and 50th day (Fig. 2f). The size of the developing tumor was decreased with the increasing dose and time of AETE, and a maximum decrease of tumor size compared to tumor control animals was noticed on the 50th day of AETE treatment at a dose of 400 mg/kg BW (Fig. 2f). The anatomical appearance of tumor-bearing animals and their contrasting variation on tumor size upon AETE administration clearly indicated the potential of AETE towards tumor regression.
AETE administration led to the restoration of tissue architecture in EAC-induced tumor-bearing mice
Tissue histology was done in liver and tumor tissues (thigh muscles) in control, tumor control, MMC, and AETE treated mice at two different time points, i.e., at 30th day and 50th day. Histological section of liver and thigh tissues of control mice showed normal tissue architecture (Fig. 3). In thigh tissues, we have seen the normal structure of skeletal muscle fibers containing multinucleated cells mostly located in the periphery of the elongated muscle cells [Fig. 3A (a-b), (k-l)]. In the tumor control group, the tissue structure of muscle fibers got disrupted, and highly nuclear-stained cells were seen. The proliferation of EAC cells and their infiltration into the inoculation site resulted in highly nuclear-stained cells [Fig. 3A (c-d), (m-n)]. Treatment of MMC and AETE re-established the disordered structure of thigh tissue in a dose and time-dependent way. On the 30th day, MMC and AETE treatment (200 mg/kg BW) did not elicit marked restoration of thigh structures. However, AETE exposure with a dose of 400 mg/kg BW for 30 days showed marked restoration of tissue architecture of thigh muscles. However, a more prominent result was observed on the 50th day of AETE treatment. On the 50th day, AETE treatment (200 mg/kg BW) showed a gradual decline of tumor masses and infiltrated cells [Fig. 3A (q-r)]. On the 50th day of AETE treatment (400 mg/kg BW), histological sections of thigh muscles [Fig. 3A (s-t)] showed more remarkable results, and EAC-induced abolished tissue got re-established, showing tissue organization similar to the control group. Similarly, darkly nuclear-stained cells were also observed in liver tissues, indicating that EAC cells may have invaded other organs [Fig. 3B (c-d), (m-n)]. However, AETE treatment (200 mg/kg BW and 400 mg/kg BW) for 30 days and 50 days showed restored tissue architecture and a lack of infiltrated cells. Upon treatment with AETE (400 mg/kg BW) for 50 days, normal tissue organization with a large and round nucleus [Fig. 3B(s-t)] similar to control group animals was observed [Fig. 3B (a-b), (k-l)].
AETE administration does not produce any toxicity in normal mice
To assess the toxicity of AETE in normal mice (no tumor-induced), we have analyzed liver and kidney function tests along with hematological parameters. The BW of AETE treated and untreated animals also analyzed, and no statistically significant difference was observed in BW change of AETE treated and untreated mice (Fig. 4a). We also noticed similar levels of ALT, AST, Urea, and Uric acid in the 14th -day blood plasma test [Fig. 4(e-h)] indicating that AETE has no toxicity in the liver and kidney. The RBC count was also not significantly different in AETE treated group (Fig. 4d); however, the Hb content and WBC count were slightly increased in AETE treated animals [Fig. 4(b-c)].
AETE treatment causes cytotoxicity to EAC and HEp-2 cells
EAC and HEp-2 cells were exposed to increasing concentrations of AETE (0.05 0.1, 0.25, 0.5, and 1 mg/ml) for 48 h and 72 h to assess the AETE induced cytotoxicity. Trypan blue dye exclusion assay showed a dose and time-dependent decrease in cell proliferation in EAC and HEp-2 cells for both 48 h and 72 h [Fig. 5(a-b)]. MTT assay was also performed in EAC and HEp-2 cells, and similar results were observed. Dose and time-dependent decrease in cell viability in both the tested cell lines [Fig. 5 (c-d)] was observed. As assessed by MTT assay, the highest decrease in cell viability for 48 h in the present study was observed in EAC cells upon exposure to 1 mg/ml of AETE, and only 6.52 ± 2.42% (p < 0.001 vs. control) cells were survived (Fig. 5c) whereas, in HEp-2 cells, the cell viability was 16.04 ± 4.03% (p < 0.001 vs. control). Similarly, at 72 h exposure, only 2.32 ± 1.11% (p < 0.001 vs. control) and 8.41 ± 3.39% (p < 0.001 vs. control) of cells were found to be survived in EAC and HEp-2 cells, respectively upon exposure to AETE with the maximum dose (1 mg/ml) (Fig. 5d). The IC50 (50% inhibitory concentration) values of AETE in EAC and HEp-2 cells were calculated at 48 h and 72 h and represented in Table 3.
LDH release assay was done in EAC and HEp-2 cells following AETE exposure (0.05, 0.1, 0.25, 0.5, and 1 mg/ml) to establish the cellular integrity of the cells. In the LDH release assay, we found a dose and time-dependent increase in LDH release following AETE treatment. At 48 h exposure, the maximum LDH release was observed in EAC cells (94.08 ± 2.65%, p < 0.001 vs. control) upon exposure to 1 mg/ml of AETE (Fig. 5e) whereas, in HEp-2 cells, the LDH release was 88.93 ± 4.29% (p < 0.001 vs. control). Similarly, at 72 h, upon exposure to the maximum dose of AETE (1 mg/ml), the LDH release in EAC and HEp-2 cells were 93.12 ± 6.58% (p < 0.001) and 95.38% (p < 0.001), respectively as compared to their respective control (Fig. 5f).
AETE exposure reduces the colony formation in EAC cells
AETE exposure to EAC cells showed a significant decrease in the colony number formation in a dose and time-dependent manner. Upon exposure to 0.2 mg/ml of AETE for 24 h, the number of colonies decreased to 45.35 ± 6.79% (p < 0.001) compared to control [Fig. 6 (a-b)]. Similarly, at 48 h of exposure with high dose (0.2 mg/ml) decreased ~ 85–90% of colonies in EAC cells, and it was found to be 11.33 ± 1.74% (p < 0.001) as compared to control [Fig. 6 (c-d)]. Therefore, in this study, we observed that AETE treatment led to dose and time-dependent inhibition in forming colonies in EAC cells compared to untreated control.
AETE exposure to EAC cells causes cell death without cell cycle arrest
EAC cells were exposed to AETE (0.05, 0.1, and 0.2 mg/ml) for 24 h and 48 h to investigate the potential of AETE in cell cycle arrest. Following AETE exposure, the percentage of cells in each stage of the cell cycle (G1, S, and G2/M phase) was analyzed. Results showed that AETE treatment does not induce cell cycle arrest in EAC cells. However, a dose-dependent increase of cells in the Sub G1 phase was observed upon AETE exposure. In the present study, AETE exposure at a dose of 0.2 mg/ml for 24 h showed a significant increase in Sub G1 phase cells (24.03 ± 1.75%, p < 0.001) as compared to control (4.70 ± 0.37%) (Fig. 7b). At the 48 h treatment period, we observed similar results of the dose-dependent accumulation of Sub G1 cells upon AETE exposure with the maximum increase of 22.55 ± 1.18% (p < 0.001 vs. control) in 0.2 mg/ml AETE treatment group (Fig. 7d).
AETE exposure causes apoptosis in EAC cells
The extent of apoptosis following AETE exposure was assessed in annexin V-FITC, and PI stained EAC cells. In the present study, we observed that AETE treatment for 24 h and 48 h led to apoptosis in EAC cells. At 24 h exposure, the incidence of early apoptotic cells, late apoptotic cells, and necrotic cells in the control group was 1.93 ± 0.19%, 3.07 ± 0.14%, and 5.73 ± 0.27%, respectively [Fig. 8(a-b)]. AETE treatment led to an increase in the incidence of apoptotic cells significantly. The highest increase in apoptotic cell types in our study was observed in 0.2 mg/ml of AETE treated cells, and the incidence of early apoptotic cells, late apoptotic cells, and necrotic cells were recorded as 4.17 ± 0.19% (p < 0.01), 5.57 ± 0.19% (p < 0.001) and 6.50 ± 0.27% respectively [Fig. 8(a-b)] as compared to their respective control. The AETE mediated induction of apoptosis showed a time-dependent effect in EAC cells. When cells were exposed to AETE for 48 h, the incidence of apoptotic cell subtypes was drastically increased compared to 24 h treatment. At 48 h, the percentage of early apoptotic, late apoptotic, and necrotic cell types in the control group were 2.40 ± 0.41%, 5.37 ± 1.30%, and 13.47 ± 2.62%, respectively [Fig. 8(c-d)]. However, AETE exposure (0.2 mg/ml) led to significant increase in apoptosis and 24.17 ± 0.45% (p < 0.001), 15.83 ± 0.57% (p < 0.001) and 10.30 ± 0.56% of cells with early apoptosis, late apoptosis and necrosis respectively was observed (Fig. 8d).
AETE exposure in EAC cells induced the depolarization of mitochondrial membrane potential (Δψm)
Mitochondrial membrane potential assay was performed in EAC cells for 24 h and 48 h exposure to understand the mechanism behind AETE induced apoptosis. We have employed the JC-1 staining method in EAC cells following AETE exposure to detect cytosolic cytochrome c levels. Upon AETE treatment, a dose and time-dependent increase in cytochrome c level were observed. At 24 h exposure time, AETE treatment (0.2 mg/ml) led to more than two-fold increase in depolarized cells (16.27 ± 0.83; p < 0.001) as compared to control (7.10 ± 0.59) (Fig. 9b). Upon increase in treatment time, the percentage of depolarized cells upon AETE exposure was also increased several-fold. At 48 h, the AETE treatment increased (~ three-fold) the incidence of the depolarised cell (20.63 ± 1.53, p < 0.01) compared to control (6.77 ± 0.40) (Fig. 9d). The several-fold increase in cells with changed mitochondrial membrane potential in the present study; suggests the potential role of AETE in mitochondria-mediated apoptosis in EAC cells.