Extract of Tagetes Erecta Could Be Used as a Potential Drug Candidate Against Cancer: A Study on the Anticancer Ecacy of Medicinal Plants Involving in Vitro and in Vivo Approach

Globally, the burden of cancer is increasing consistently. Modern cancer therapies include lots of toxicity in the non-targeted organs reducing the life expectancy of the patients. Therefore, the development of safer alternative medicines with less toxicity and high ecacy is of immense importance. The present study was designed to evaluate the anticancer activity of a medicinal plant, “Tagetes erecta” (TE), in established cancer cell lines in vitro and in animal models in vivo. GC-MS analysis was performed that revealed hexadecanoic acid, Linolenic acid, Quinic acid, 2,3- dihydrobenzofuran (Coumaran), and β-stigmasterol as major bioactive compounds in TE leaves. Aqueous extract of Tagetes erecta (AETE) treatment potentially reduced the tumor weight (TW) and tumor volume (TV) and increased the life span in EAC-induced tumor-bearing Swiss albino mice. Side effect analysis conrmed the lack of toxicity of AETE to non-targeted organs in normal Swiss albino mice. Studies in cancer cell lines indicated dose and time-dependent cytotoxicity in Human laryngeal carcinoma (HEp-2) and Ehrlich ascites carcinoma (EAC) cells. Flow cytometric analysis established signicant induction of apoptosis in EAC cells without arresting the cell cycle. In addition, AETE treatment led to a signicant increase in cells with depolarised mitochondrial membrane potential. The present study indicated that AETE potentially inhibits tumor progression without disturbing normal body physiology. Thus, we conclude that AETE can be used as a potential therapeutic agent against cancer.


Introduction
Cancer is considered as a severe public health problem and the leading cause of worldwide morbidity and mortality [1,2]. It is characterized by rapid cellular proliferation evading the cell cycle checkpoints. Despite the rapid development in cancer biology, the incidences of cancer and its related mortality rate are increasing consistently. Recently, International Agency for Research on Cancer produced GLOBOCAN 2020 estimate on the worldwide cancer burden and reported that the new cases of cancer are raised to 19.3 million, out of which 10 million cancer-related deaths have occurred [3]. The increasing mortality rate in cancer is primarily due to the less effective diagnostic approach and the side effects of modern cancer treatment approaches.
The primary diagnostic approaches of cancer include chemotherapy, radiation therapy, surgery, targeted therapy, and immunotherapy [4,5]. Unfortunately, these diagnostic strategies failed to show the anticipated outcome. Moreover, these treatments are also associated with the recurrence of cancer, toxicity to non-targeted organs, and drug resistance [6]. Therefore, scientists are constantly searching for novel and promising anticancer therapeutics of enhanced e cacy and lesser toxicity to overcome this deadly disease.
For decades, medicinal plants have always been utilized to treat various diseases, including cancer, because of their wide range of biological and therapeutic potential. The tremendous therapeutic property of the medicinal plants is attributed to the bioactive compounds present therein. Therefore, scientists worldwide are investigating the medicinal properties of plants to develop novel drug molecules with improved e cacy. Furthermore, the use of plants and plant-based products in cancer management may reduce the unwanted toxicity associated with conventional chemotherapeutic drugs. In the present, we investigated the anticancer activity of a commonly used medicinal plant, Tagetes erecta (TE), against EAC-induced solid tumor in mice in vivo and in cell lines in vitro.
Tagetes erecta L., commonly known as marigold, belongs to the family Asteraceae. TE is exploited to treat bronchitis, cold, rheumatic pain, headache, ulcers, and respiratory diseases [7]. TE is also being used to treat diabetes mellitus [8,9]. Different parts of TE have been reported to possess various pharmacological properties. In scienti c literature, the wound healing activity of the leaves of TE is well described [10][11][12]. In addition, the roots of TE have antimicrobial and antiplasmodial effects [13]. Pharmacologically, TE exhibits antioxidant, anti-in ammatory, anti-diabetic, anti-depressant, antibacterial, and insecticidal activity [9,14] that can be attributed to the potential bioactive compounds present in the plant. However, only a few studies of the anticancer potential of TE are available in cell lines only [15,16]. Reporting of any drug agent or compound's anticancer potential, it's testing in cell line only is inadequate and must go for further preclinical and clinical experimentation. Therefore, this study was carried out to evaluate the potential of aqueous extract of Tagetes erecta (AETE) towards tumor regression in EAC-induced solid tumor-bearing mice in vivo and in HEp-2 and EAC cell lines in vitro. HEp-2 is a human laryngeal carcinoma cell with a high proliferative rate of the cell cycle. Ehrlich ascites carcinoma (EAC) is an undifferentiated breast adenocarcinoma cell with high transplant ability and rapid cellular proliferation with 100% malignancy [17]. This study hypothesized that AETE might exhibit potential cytotoxic activity in cancer cells and resulting retardation of tumor growth in a model animal.
In our in vivo studies, we observed that AETE treatment potentially reduces the tumor growth by reducing tumor weight (TW) and tumor volume (TV) in EAC-induced solid tumor-bearing mice as compared to tumor control and Mitomycin C (MMC) treated group. In this study, MMC is considered a positive control. MMC is a standard anticancer drug and induces cytotoxicity in cells underexposed. AETE administration in solid tumor-bearing mice also showed a substantial increase in life span (ILS) of tumor-bearing animals compared to the tumor control group. In cell culture studies, AETE exposure showed dose and time-dependent cell death in HEp-2 and EAC cells and induced apoptosis by changing mitochondrial membrane potential.
Collection and identi cation of the plant Fresh and healthy leaves of TE were collected directly from the plants of Southern Assam, India. Geographically, the latitude and longitude of the plant collection site are 24°47 01N and 92°47 50E respectively on degree minute second (DMS). A herbarium of the plant specimen was prepared and identi ed, and authenticated by the taxonomist at Botanical Survey of India, Shillong, Meghalaya, India.
Preparation of aqueous extract of Tagetes erecta (AETE) The collected leaves of TE were washed with double distilled water twice and were air-dried. The dried leaves were then mechanically ground into a ne powder using a motor grinder. To extract the chemical ingredient, 25 g of the ground powder was taken in the extractor of the soxhlet apparatus (BOROSIL, India), and 250 ml of double-distilled water was poured into the collection ask. The extraction procedure was run up to 6-8 h at 37 ± 1°C. The soluble extract was then ltered using Whatman No. 1 lter paper until clear and then lyophilized. The percentage of yield of the extract was 24.6%. The lyophilized extract was stored in a refrigerator at -20°C until use but not for more than one month. The extract was dissolved in double-distilled water before use.

Gas chromatography and mass spectrometry (GC-MS) analysis
The procedure of extraction of AETE for GC-MS analysis was the same as mentioned above. GC-MS analysis of the plant extract was carried out using GC-MS QP2010 to characterize the chemical composition of AETE. The GC-MS ultra system is connected to an Elite 5MS (5% Diphenyl in diethyl polysiloxane) attached with a capillary column. Helium (99.99%) was used as a carrier gas with a constant ow of 1.21 ml/min in split mode. Both injector and detector temperature was set to 260°C and 270°C, respectively, while the column oven temperature was adjusted at 50°C. After 2 min, the column oven temperature was raised to 250°C at a constant rate of 7°C/min and again from 250°C to 280°C at a constant speed of 15°C/min with a holding time of 16 min. The whole process runs for 46.56 min. The peaks obtained in the chromatogram were interpreted using the National Institute of Standard and Technology (NIST, USA) database to identify the bioactive compounds.
In vivo experiments Animals Swiss albino mice of Balb/c strain (25-30 g body weight, 6-8 weeks old) were procured from Pasteur Institute, Shilling, Meghalaya, India. The animals were kept in polypropylene cages (TARSONS, India) and acclimatized to laboratory conditions for 1-week prior to experimentation. Animals were housed at a maintained temperature of 25 ± 5°C and 12 h light and 12 h dark photoperiod cycles. Animals were fed with standard food pellets (Mice Maintenance Feed, LAB-061119) purchased from Hindustan Animal Feeds, Gujarat, India, and water ad libitum throughout the experiment.

Ethical permission
The experimental animals were maintained and housed in the animal house of the Department of Life Science and Bioinformatics following the strict guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The present study has ethical approval (Reference No. IEC/AUS/2015-030 dt. 4/9/15) of the Institutional Ethics Committee (IEC) of Assam University, Silchar.

Preparation of cell line and tumor induction
Ehrlich ascites carcinoma (EAC) cells (1х10 6 cells/animal) were injected directly into the peritoneal cavity of a mouse and allowed to multiply. After successful multiplication, the cells were withdrawn, diluted with PBS, and re-injected (1х10 6 cells/animal) into the thigh muscles of experimental animals as per our earlier publication [2] to develop a solid tumor.

Study design
One hundred Swiss albino mice (6-8 weeks old, weighing 25-30 g) were divided into two clusters (50 animals in each set) for endpoint evaluation on the 30th and 50th days of AETE treatment. In each cluster, 10 animals (5 male and 5 female) were kept for the control group, and the remaining 40 animals were inoculated with EAC cells to develop a solid tumor. After the successful development of solid tumors, these 40 animals were randomly divided into 4 groups containing 10 animals (n = 10, 5 male, and 5 female) in each group. The different treatment groups for evaluating the anticancer potential of AETE were as follows- In a previous report, MMC treatment at a dose of 2 mg/kg BW per week showed the highest decrease of tumor weight (TW) in EAC-induced solid tumor-bearing animals amongst the doses treated (0.5, 1 and 2 mg/kg BW) [18]. Therefore, in the present study, we have selected the MMC dose of 2 mg/kg BW per week to compare the results of positive control drugs with AETE treatment.

Dose and treatment of AETE for in vivo study
The dose for AETE was selected based on the limit test at 2000 mg/kg as per the OECD guidelines-425 (OECD Test Guideline 425) [19]. In the limit test study, the animals were fasted from food only prior to dosing, and based on the 3-h fasted BW, one animal was treated with 2000 mg/kg AETE. As the animal was survived, four more animals were again treated with AETE in the same manner and observed for the next 14 days. No mortality was observed, and all ve animals survived. Thus, the LD 50 for AETE was assumed to be more than 2000 mg/kg BW. Based on this observation, in this study, we have selected two doses for AETE (200 mg/kg BW and 400 mg/kg BW) as the chosen dose could not produce toxicity due to a greater LD 50 value than 2000 mg/kg BW. However, to con rm AETE mediated toxicity, if any, we have performed side effect analysis in normal mice following AETE treatment (400 mg/kg BW) for 14 days.
Evaluation of the anticancer potential of AETE in mice The anticancer potential of AETE in mice was assessed by analyzing the tumor volume (TV), tumor weight (TW), and body weight (BW). TV and BW were measured on every alternative day throughout the treatment period.

Measurement of body weight (BW)
The BW of the experimental animals was recorded on each alternative day, and it was analyzed at twotime points (at the 30th and 50th day of AETE treatment). The effect of AETE treatment in control and EAC-induced solid tumor-bearing animals was analyzed for initial, nal, net BW and percentage of change in BW from initial BW. The net BW and change in BW from initial weight was calculated as per the following equations-

Measurement of tumor volume (TV)
TV of the tumor-bearing animals was measured on every alternative day throughout the treatment period by using a vernier caliper (Mitutoyo 530 Series Vernier Caliper). The TV of the experimental animals was calculated by using the equation V = 0.5 ab 2 , where 'V' is the volume and 'a' and 'b' is the major and minor diameter, respectively [2,20].

Measurement of tumor weight (TW)
After the 30th and 50th days of AETE treatment, animals were sacri ced by cervical dislocation; tumor tissues were excised and weigh out to assess the effect of AETE on TW. The results of AETE mediated TW reduction in EAC-induced tumor-bearing animals was shown in Fig. 2d.

Assessment of survival time in EAC-induced solid tumor-bearing mice
The effect of AETE on life span was assessed in Swiss albino mice. Forty experimental animals were inoculated with EAC cells (1х10 6 cells/animal) and left for tumor development. On the 12th day of EAC inoculation, a solid tumor was developed. These 40 EAC-induced solid tumor-bearing mice of both sexes were randomly divided into four groups containing 10 animals in each group (n = 10, 5 male, and 5 female). Group 1 animals were kept without any treatment and termed as tumor control. Group 2 (MMC) animals were treated with MMC (2 mg/kg BW per week). Group 3 and Group 4 animals were exposed to AETE at a dose of 200 mg/kg BW (AETE 200) and 400 mg/kg BW (AETE 400) respectively till the 50th day. All the experimental animals were kept under surveillance for the entire lifetime, and the death pattern was recorded. The effect of AETE on life span was analyzed by calculating the increase in life span (ILS) compared to tumor control animals. The ILS was calculated using the following formulae-Tissue histology of liver and tumor tissues Histological evaluation in liver and tumor tissues (thigh muscles) was done to assess the changes in tissue structure. The liver and tumor tissues from control, tumor control, MMC, and AETE treated animals of both 30th and 50th days of treatment were extracted out, immediately xed in 10% formalin, and were processed for histology. Brie y, xed tissues were properly washed with water to remove the xatives, dehydrated with graded alcohol, and then embedded in para n wax. The para n-embedded tissues were then sectioned into 5µm thickness in a microtome. Finally, the sectioned tissues were depara nized, rehydrated, and stained with hematoxylin and eosin (H&E). The slides were observed in a light microscope (Leica DMLS, Leica, Wetzer, Germany) at different magni cations, and photographs were acquired.

Analysis of the adverse effect of AETE in normal mice
The toxicity pro le of AETE was assessed in normal animals. Twenty Swiss albino mice of both sexes were randomly divided into two groups containing 10 animals in each group (n = 10). Group 1 (control) animals were kept without treatment, while Group 2 (AETE 400) animals were treated with AETE at a dose of 400 mg/kg BW for 14 days. The BW of the experimental animals was recorded on each alternative day. Liver and kidney functions test (ALT; Alanine aminotransferase, AST; Aspartate aminotransferase, Urea, and Uric acid) was performed in the blood plasma of control and treated animals. Hematological parameters (hemoglobin content, WBC, and RBC number count) were also evaluated to assess the toxicity of AETE if any.

In vitro experiments
Cell culture EAC and HEp-2 cells were procured from National Centre for Cell Sciences (NCCS, Pune, India). Both the cells were cultured in a minimum essential medium (MEM), constituting 10% fetal bovine serum (FBS), 100 U antimycotic solutions (Himedia). The culture condition was maintained at 37°C in a humidi ed environment with a continuous supplement of 5% CO 2 .
Tryan blue dye exclusion assay AETE induced cytotoxicity was assessed using trypan blue dye exclusion assay in EAC and HEp-2 cells.
Brie y, EAC and HEp-2 cells (1х10 4 cells/well) were seeded in six-well plates. After attaining ~ 70% con uency, cells were exposed to different concentration of AETE (0.05, 0.1, 0.25, 0.5 and 1 mg/ml) for 48 h and 72 h. Cells without any treatment were considered as control. After treatment, cells were harvested, and numbers of live cells were counted at least three times per treatment level. All the treatments were performed in triplicates (n = 3). The IC 50 value (50% inhibitory concentration) of AETE for 48 h and 72 h for EAC and HEp-2 cells were determined and presented in Table 3.

MTT assay
MTT [3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay was done in EAC and HEp-2 cells following AETE exposure for 48 h and 72 h. EAC and HEp-2 cells (1х10 4 cells/well) were seeded in 96 well culture plates. As soon as the cells attained ~ 70% con uency, they were treated with increasing concentration of AETE (0.05, 0.1, 0.25, 0.5 and 1 mg/ml) for 48 h and 72 h in triplicates (n = 3). Cells without treatment were considered as control. After 48 h and 72 h exposure, media was aspirated carefully, and fresh media was added to it. A 20 µl of MTT solution (5 mg/ml) was added to the media and incubated in a CO 2 incubator for another 3 h. The purple-colored formazan crystals formed were dissolved using DMSO. Finally, the optical density was read at 570 nm using a microplate reader.
LDH release assay LDH (Lactate dehydrogenase) release assay was done following the protocol of Korzeniewski and Callewaert [21] with modi cations of Kavitha et al. [22]. Brie y, EAC and HEp-2 cells (1х10 4 cells/well) were seeded in 96 well plates. At ~ 70% con uency, the cells were exposed to different concentrations of AETE (0.05, 0.1, 0.25, 0.5 and 1 mg/ml) for 48 h and 72 h. Cells without treatment were considered as control. All the treatments were performed in triplicates (n = 3). After treatment, the LDH level released in media and cell lysate was measured by acquiring the OD (490 nm) using a microplate reader. The percentage of LDH release following treatment was calculated using the following equation-

Colony formation assay
Colony formation assay was performed in EAC cells as per the standard protocol [23,24]. Brie y, EAC cells (1000 cells/well) were seeded in 6 well plates in triplicates (n = 3). As the cells attached to the culture disk, they were treated with AETE (0.05, 0.1 and 0.2 mg/ml) for 24 h and 48 h. After exposure, the media was aspirated out carefully, and fresh media was added to each well and kept in a CO 2 incubator. Cells without treatment were considered as control. The media was being replaced by fresh media whenever necessary. As the cells of control plates formed colonies of su cient size, the media from all the wells were removed, washed with PBS carefully, and xed with 6% glutaraldehyde. Then, the cells were stained with 0.5% crystal violet. The numbers of colonies formed in control and AETE treatment groups were counted. A group of cells considered as a colony if there are more than 50 cells at least.

Cell cycle assay
Cell cycle assay was studied in EAC cells. EAC cells (1х10 4 cells/ml) were seeded in a six-well culture plate. As the cells attained ~ 70% con uence, cells were exposed to AETE (0.05, 0.1 and 0.2 mg/ml) for 24 h and 48 h. After treatment, cells were harvested and treated with 0.1% citrate buffer containing NP40 (0.3 µl/ml). Then, the cells were treated with PI/RNase solution (BD Biosciences, product code: 550825) and incubated for 20 min in dark conditions at 4°C. The cells were vortexed properly, and cellular DNA content was detected using a ow cytometer (Accuri, BD Biosciences, USA). The experiment was done in triplicates (n = 3), and from each sample, 15,000 cells were recorded and analyzed.

Apoptosis assay
To assess the AETE induced apoptosis in EAC cells, 1х10 4 cells/ml were seeded in six-well culture plates. After attaining ~ 70% con uence, cells were treated with AETE (0.05, 0.1 and 0.2 mg/ml) for 24 h and 48 h. AETE induced the extent of apoptosis by using FITC Annexin V Detection Kit as per the manufacturer's instruction (BD Biosciences, USA). Brie y, harvested cells were rinsed with ice-cold PBS, stained with annexin V-FITC/PI conjugate (BD Biosciences), and incubated at 37°C for 15 min. Then, the cells were mixed with binding buffer (400 µl/sample), and 15,000 cells per sample were analyzed using a ow cytometer (Accuri, BD Biosciences, USA).
Mitochondrial membrane potential (Δψm) assay Mitochondrial membrane potential was analyzed in EAC cells following AETE treatment for 24 h and 48 h. Brie y, EAC cells (1х10 4 cells/ml) were seeded into six-well culture plates. As the cells attained ~ 70% con uency, they were exposed to different concentrations of AETE (0.05, 0.1 and 0.2 mg/ml) for 24 h and 48 h. Then, the cells were harvested and centrifuged at 1500 rpm for 5 min. Next, the pelleted cells were re-suspended and mixed with JC-1 dye (5.5 , 6, 6 -tetrachloro-1, 1 , 3, 3 -tetraethylbenzidazolylcarbocyanine iodide, BD Biosciences) and incubated at 37°C for 15 min followed by washing with 1X assay buffer (BD Biosciences) for 2-3 min. Again, the cells were spun at 1500 rpm for 5 min, and the pellet was re-suspended in 0.5 ml of fresh 1X buffer. Finally, the cells were vortexed properly, and 15,000 cells/sample were analyzed in a ow cytometer to analyze the depolarized cells.

Statistical analysis
One-way analysis of variance (ANOVA) was performed to test the signi cance level among the groups, followed by Tukey post-hoc test for multiple comparisons. For side effect analysis, Students 't' test was performed to assess the signi cant differences. Variances were considered to be signi cant at α error probability value of 0.05. All the values in the manuscript were represented as mean ± SD. All the analysis was performed in Graph Pad Prism software version 7.0 (San Diego, California, USA). Survival data were analyzed in IBM SPSS software version 21 for Windows (Armonk, NY: IBM Corp.)

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  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 timedependent 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 signi cant 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) (       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). Signi cant 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 bers containing multinucleated cells mostly located in the periphery of the elongated muscle 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 signi cant 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 signi cantly 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)]. 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) Table 3.

AETE treatment causes cytotoxicity to EAC and
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 timedependent 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).  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.  (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 signi cantly. 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 signi cant 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.

Discussion
The development of new therapeutics with less toxicity and side effects is the major challenging task in cancer biology. There was a well-built correlation between the higher intake of vegetables and plantbased products with the reduced risk of cancer incidences. The present study aimed to investigate the cancer therapeutic potential of T. erecta, a well-known medicinal plant, against the established cell lines and in EAC-induced solid tumor-bearing mice. The anticancer activity of TE has been reported by scientists using in vitro assays in cancer cell lines only [15,16], but the in vivo anticancer potential of TE in mice has not been studied so far. Testing a new therapeutic agent in both in vitro and in vivo models remains important for investigational new therapies. Animal tumor models preserve cell-cell interactions' characteristics and enable pharmacokinetic and toxicity assessment of new investigational compounds.
In the present study, we have reported the antitumor potential of AETE in established cancer cell lines and EAC-induced solid tumors-bearing Swiss albino mice. Our animal model study revealed dose, and timedependent decreased in TW and TV in tumor-bearing animals. The survival time of tumor-bearing experimental animals was also increased in increasing dose and time. Similar work has been carried out in the mice allograft model by taking different plant extracts or isolated plant products. In a study, crude extracts of Eucalyptus camaldulensis decreased the TW showing its antitumor activity in EAC-induced solid tumor-bearing mice [25]. Previously, we also reported the potent anticancer activity of aqueous extract of Moringa oleifera (AEMO) in EAC-induced tumor-bearing mice [2]. In a study, methanol extract from the leaves of Trema Orientalis also reduced EAC cell growth in experimental mice [26]. Reports are available that the isolated active ingredients also exhibit anticancer activities in the mice test system. A study by Srivastava et al. [27] reported that quercetin treatment showed the potential role in TV reduction in EAC-induced tumor-bearing animals. At the same time, Thomas et al. [28] also showed a remarkable reduction of TV and TW when the experimental animals were exposed to extracts of Vernonia condensate. Our results of AETE mediated tumor reduction in EAC-induced tumor-bearing animals were also in concordance with the study of Noaman et al. [29], where they observed dose and time-dependent effects on TW reduction in arabinoxylan rice bran (MGN-3/biobran) treated groups.
Our study indicated the increase in survival time of tumor-bearing animals upon AETE administration.
The life span of tumor-bearing animals was increased ~ 3 fold and ~ 5 fold when administered with AETE at a dose of 200 mg/kg BW and 400 mg/kg BW, respectively. Upon administration of AETE with a high dose (400 mg/kg BW), at least 50% of the animals were survived more than 280 days, whereas, in tumor control, all the animals have died within 62 days. The histopathological evaluation of liver and thigh tissues showed restoration of disrupted tissue structure upon AETE administration. The thigh tissues in the control group had normal architecture with elongated muscle bers and a nucleus. In the tumor control group, the architecture of muscle tissues was disrupted. Upon administering AETE, the muscle tissue structures were recovered, and in the 400 mg/kg BW treatment group, the abolished muscle tissue morphology was re-established maximally. The EAC cell has malignant properties and can invade other organs of the EAC-bearing animals. The liver histology in the control group showed the normal structure of hepatocytes with a large and round nucleus. However, in EAC solid tumor-bearing animals, darkly stained in ltrated EAC cells (sinusoidal in ltration) were clearly visible. EAC cells have vast potential to metastasize to other organs like the liver, lungs, kidney, spleen, etc. AETE treatment signi cantly reduced the altered hepatic morphology. Upon treatment with 400 mg/kg BW of AETE for the 50th day, reestablishes the liver morphology by reducing the metastasized EAC cells. Based on the above observations, the present study indicated that AETE is a potent cytotoxic agent to HEp-2 and EAC cells and inhibits EAC cells' proliferative potential, leading to an increase in life span of tumor-bearing mice.
Further, the toxicity pro ling of AETE did not show any signi cant alternations in AST, ALT, Urea, and Uric acid levels in the 14th -day serum test of AETE treated mice [ Fig. 4 (e-h)]. The BW and RBC count also did not show any signi cant changes [ Fig. 4(a, d)]. However, the Hb content and WBC count showed little variation in AETE treated mice compared to control [ Fig. 4 (b-c)]. Thus, the present study reports that AETE is a good and safe candidate for developing a new cancer therapeutic drug that can inhibit cancer progression without affecting the normal body metabolism.
Results of in vitro studies showed the cytotoxic potential of AETE in EAC and HEp-2 cell lines. Previous studies also reported the cytotoxic effect of T. erecta in a variety of cell lines. A study said that ethanolic extract of T. erecta roots showed extensive cytotoxicity in the prostate (PC-t3) and HeLa cancer cell lines [30]. The essential oil extracted from T. minuta, a closely related species of T. erecta, showed induction of cytotoxicity in human promyelocytic leukemia (HL-60, NB4) and EAC cell lines [31]. Not only leaves but the other parts of T. erecta are also being investigated for their anticancer activity. The ethanolic and ethyl acetate extract from the owers of T. erecta has shown cytotoxicity to lung (H460) and colon cancer (Caco-2) cell lines [32]. It is reported that essential oils extracted from T. erecta showed signi cant cell death in a variety of cell lines like colon adenocarcinoma (HT29), human glioblastoma (MO59J, U343, U251), human hepatocellular carcinoma (HepG2), murine melanoma (B16F10), human cervical adenocarcinoma (HeLa) and human breast adenocarcinoma (MCF-7) cells [7]. Therefore, the dose and time-dependent cytotoxicity of AETE in EAC and HEp-2 cells in our study and above reports also suggests that irrespective of the cancer type, AETE could induce cell death, as shown by three independent assays in the present study.
In the present study, AETE exposure inhibits the formation of colonies in EAC cells in a dose and timedependent way, suggesting the potential of AETE towards regression in the tumorogenic potential of EAC. Further, cell cycle assay analysis did not show cell cycle arrest upon treatment with AETE. However, dosedependent accumulation of cells at the sub G1 phase (Fig. 7) indicated the apoptosis-inducing potential of AETE. The dose-dependent increment of apoptosis was further validated by annexin-V FITC assay that con rmed the dose and time-dependent increment of annexin-V FITC positive cells (Fig. 8). Annexin-V FITC is a Ca 2+ dependent phospholipid-binding protein having a high a nity to bind to  [2,33]. Further, to understand the mechanism of AETE induced apoptosis in the EAC cell line, we have performed mitochondrial membrane potential (Δψm) in EAC cells by using JC-1 dye. JC-1 is a potent dye with a high binding a nity upon the release of cytochrome c into the cytoplasm.
Depolarization of mitochondrial membrane leads to the release of cytochrome c from its membrane to the cytoplasm. This phenomenon leads to the shifting of red uorescence to green uorescence. Upon treatment with AETE, we observed dose and time-dependent increase of green uorescence, indicating dose and time-dependent increase of cells with depolarized mitochondrial membrane potential (Fig. 9).
Cytochrome c is released from the mitochondrial membrane in early apoptotic cells due to activation of proapoptotic factors [34]. In the presence of cytochrome c, activating apoptotic protease activating factor-1 (apaf-1), initiates apoptosome complex formation [35,36]. Apoptosome complex then activates Ca 2+ dependent serine proteases of the intrinsic pathway (caspase-9), which activates the downstream signaling cascade and activates caspase-3 leading to programmed cell death [37]. Therefore, the increased percentage of apoptotic cells following AETE treatment in the present study might be due to the activation intrinsic pathway of apoptosis. However, it needs further validation by designing translational studies.
The cancer prevention potential of the plants is attributed to a range of bioactive compounds present in plants. GC-MS analysis of AETE showed the presence of several bioactive phytochemicals. Among them, the major active ingredients were Heaxadecanoic 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. A previous study reported that palmitic acid causes cytotoxicity in human leukemic (MOLT-4) and HCT-116 cells at microgram level and potent antitumor activity in mice [38,39]. There are also reports suggesting the anti-in ammatory activity of palmitic acid [40] and linolenic acid [41]. Quinic acid, a major bioactive compound in TE leaves, has extensive antioxidant, antiin ammatory, and antimutagenic potential [42,43]. A recent study reported the anticancer potential of quinic acid against oral cancer by modulating cyclin D1 and Akt signaling [44]. The quinic acid derivatives also played a crucial role against human colon and prostate cancer [45,46]. The derivatives of 2,3-dihydro benzofuran isolated from Polygonum barbatum showed potential anticancer activity against oral cancer (CAL-27) and lung cancer (NCI H460) cell lines by inducing apoptosis [47]. A recent study reported that β-stigmasterol induces apoptosis in ovarian cancer (ES-2 and OV-90) cells by regulating endoplasmic reticulum and mitochondria functioning [48]. Another report suggests that β-stigmasterol also suppresses angiogenesis and inhibits cholangiocarcinogenesis in mice by downregulating tumor necrosis factor α [49]. Therefore, the present study indicated that the potent anticancer e cacy of AETE in established cancer cell lines and in vivo tumor-model is due to the various bioactive compounds present in TE leaves.

Conclusion
In the present study, we observed that AETE has potential cytotoxic activity against HEp-2 and EAC cell lines. AETE exposure also leads to apoptosis by releasing cytochrome c from mitochondria. Thus, we conclude that AETE has immense potential to induce apoptosis by depolarizing the mitochondrial membrane. However, the present study is de cient in the protein expression study. Characterization of proapoptotic proteins responsible for apoptosis is the future direction of the present study that will confer a more detailed view of AETE mediated apoptosis. The in vivo results in TV and TW reduction following AETE administration clearly indicated the tumor regression potential of AETE against EAC-induced solid tumors.
Moreover, restoration of disrupted tissue morphology in tumor-bearing allograft mice and improvement in life span is another evidence of the antitumor potential of AETE. Further, side effect analysis of AETE did not show potential toxicity in normal mice. Thus, summarizing the results, we conclude that AETE has strong anticancer potential without producing toxicity in other body organs and could be utilized to develop a new therapeutic agent against cancer.

Con ict of interest statement
There is no con ict of interest to declare Author's contribution   Analysis of adverse toxicity of AETE in normal mice. Side effects for AETE administration were analyzed in normal mice upon AETE treatment for 14 days (n=10), and changes in BW (a), blood parameters (b-d), liver parameters (e-f), and kidney parameters (g-h) was done in 10 experimental animals (n=10).
Statistical analysis: student's t-test. Hb, Heamoglobin; WBC, White blood cells; RBC, Red blood cells; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase. (e-f). LDH release assay showing lactate dehydrogenase release in EAC and HEp-2 cells upon exposure to different concentrations of AETE for 48 h (e) and 72 h (f). All the experiments were performed in triplicates (n=3), and each data set in the graphs showed mean, and error bars indicate SD. Statistical analysis: One-way ANOVA. ***p <0.001, **p <0.01 and *p <0.05 vs respective control. ns; not signi cant. Effect of AETE in colony formation at 48 h of AETE exposure. All the experiments were performed in triplicates (n=3), and each data set in the graphs showed mean, and error bars indicate SD. Statistical analysis: One-way ANOVA. ***p <0.001, and *p <0.05 vs respective control.