Mussaenda macrophylla Wall. exhibit anticancer activity against Dalton’s lymphoma ascites (DLA) bearing mice via alterations of redox-homeostasis and apoptotic genes expression

Mussaenda macrophylla is a shrub widely used in Mizo traditional practice for treatment of cancer, fever, cough, ulcer and dysentery. We have previously shown the antioxidant nature of the plant. In this study, we explore the anticancer activity of the aqueous extract of M. macrophylla (MMAE) using Dalton’s lymphoma ascites (DLA) bearing mice as our model. MMAE significantly inhibited the tumor growth and increased the survival time of the tumor bearing DLA mice. MMAE significantly increased the glutathione (GSH) levels; and glutathione-s-transferase (GST) and superoxide dismutase (SOD) activities. Consistently, MMAE decreased lipid peroxidation levels in DLA mice. Reduced RBC and hemoglobin levels were significantly reversed by MMAE treatment. MMAE also lowers the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CRE) levels that were otherwise elevated in the DLA control animals. Induction of DNA damage, up-regulation of pro-apoptotic genes and down-regulation of anti-apoptotic genes in DLA bearing mice following MMAE treatment provide an insight into apoptosis based anticancer activities of M. macrophylla. Our findings demonstrate the role of the aqueous extract of M. macrophylla as a potential anticancer agent possibly targeting the apoptotic pathway.


Background
Cancer is a heterogeneous disease with multiple genotoxic and oncogenic aberrations characterized by uncontrolled proliferation, invasion and metastasis of cells (Hanahan & Weinberg, 2000). There are more than 36 major types of cancer with a striking fatality rate of 9.6 million people per year. It is a major health burden worldwide and it is expected to be the leading cause of death throughout the world in the 21st century (Bray et al., 2018;Rawla & Barsouk, 2019). Non-Hodgkin lymphoma is the 13th most frequently diagnosed cancer in the world and 11th leading cause of cancer mortality worldwide, accounting for 248,724 deaths (2.6% of the total) each year (Bray et al., 2018). Dalton's lymphoma, a murine non-Hodgkin's transplantable T-cell lymphoma, that originated at the National Cancer Institute (NCI), USA in 1947 in the thymus gland of a DBA/2 mouse. The line is then maintained by serial transplantation from mouse to mouse intraperitoneally (Chakrabarti et al., 1984). Dalton's lymphoma has served as a convenient model for studying various parameters of cancer progression, signalling mechanisms, and ultimately for screening of drugs for effective treatment (Das & Vinayak, 2014). In addition, Swiss albino mice serves as an excellent model for anticancer drug screening due to their easy accessibility and similarity with humans in terms of their genomic, anatomy and immunological system (Bernardi et al., 2002).
Of the hundreds of chemicals that have been and are being evaluated for their anticancer activities, natural compounds derived from medicinal plants offer a potential resource for development of new anticancer agent(s) due to their safety, efficacy and lesser side effects when compared with synthetic drugs (Thillaivanan & Samraj, 2014). Therapeutic drugs derived from different medicinal plants have been reported to play a crucial role as anticancer agents in various experimental models of cancer. In fact, about 60% of the currently available anticancer drugs are derived from plant sources (Kamal et al., 2014). Recent studies have demonstrated the anticancer properties of various plants such as Emilia sonchifolia (Shylesh & Padikkala, 2000), Solanum pseudocapsicum (Badami et al., 2003), Astraeus hygrometricus (Mallick et al., 2010), and Sesbania grandiflora (Laladhas et al., 2010) in Dalton's lymphoma ascites bearing mice.
Mussaenda macrophylla, locally known as Vakep, is a flowering shrub that belongs to the Rubiaceae family. It is endemic to southeast Asia and is known to occur in China, Myanmar and India (Manandhar, 2002). Traditionally, different health problems such as sour mouth, sour throat, oral infections, fever, cough, dysentery, diarrhea, indigestion, chronic ulcer, cancers and snake bites have been treated using various parts of this plant (Kim et al., 1999;Rosangkima & Jagetia, 2015). Our preliminary study revealed the presence of significant

Graphical Abstract
Page 3 of 14 Lalremruati et al. The Journal of Basic and Applied Zoology (2022) 83:6 number of phytochemicals including phenols, flavonoids, alkaloids, cardiac glycosides, saponins, steroids, tannins and terpenoids in M. macrophylla (Lalremruati et al., 2019). M. macrophylla have also been reported to show multi-pharmaceutical activities including anticoagulant, anti-inflammatory and hepatoprotective activities (Dinda et al., 2008), anti-microbial (Chowdhury et al., 2013, antioxidant (Lalremruati et al., 2019), thrombolytic  and anti-diabetic activities (Bhandari et al., 2020). Given the present state of the scientific evidence on various pharmaceutical applications of M. macrophylla, high priority research is required to objectively assess the potential anticancer activity of M. macrophylla. Therefore, the present study is carried out to investigate the anticancer activity of M. macrophylla aqueous extract in Dalton's Lymphoma Ascites (DLA) bearing Swiss albino mice.

Animals and tumor model
Swiss albino mice of both sexes (10-12 weeks old) weighing 25-30 g were selected from an inbred colony maintained under controlled conditions of temperature (23 ± 2 °C) and light (12 h of light and dark, respectively) at the Animal Care Facility, Department of Zoology, Mizoram University. The animals had free access to food and water. The animal care and handling were performed according to the guidelines of World Health Organization, Geneva, Switzerland. Dalton's lymphoma ascites (DLA) tumor has been maintained by serial intraperitoneal (i.p) transplantation of about 1 × 10 6 viable tumor cells in 10-12-weeks-old mice under aseptic condition.

Preparation of drug and mode of administration
The aqueous extract of M. macrophylla (MMAE) and doxorubicin (a standard drug) were dissolved in distilled water. Animal from each group received different dose of treatments according to body weight intraperitoneally (i.p).

Acute toxicity study
The acute toxicity study (Prieur et al., 1973) of MMAE was performed in Swiss albino mice as per the OECD guidelines 420-425. Animals of both sexes were randomly divided into four groups of ten animals each (n = 10), and treated with aliquot doses of MMAE intraperitoneally (1.2, 1.4, 1.6 and 1.8 g/kg b.wt) and monitored for mortality and toxic symptoms up to 14 days post-treatment. The LD 50 value of the MMAE was calculated using probit analysis (Miller & Tainter, 1944). The probit values were then plotted against log-doses and the dose corresponding to probit 5, i.e., 50%, was calculated. The standard error of mean (SEM) of LD 50 was calculated using the formula: where N is number of animals in each group.

Experimental design
For the assessment of survival time and weight change, the animals were randomly distributed to five equal groups (n = 6). All mice were transplanted (i.p) with 1 × 10 6 cells in 0.25 mL of PBS on day '0' . Group I was treated as the control group, which received 0.25 mL of distilled water. Group II-IV were treated (i.p) with MMAE at the dose of 50, 100 and 150 mg/kg b.wt, respectively. Group V received doxorubicin (DOX) at the dose of 0.5 mg/kg b.wt as a standard drug. After 72 h of tumor transplantation, treatment was given to each group for 7 consecutive days. Fresh experimental groups were formed as described above for the estimation of antioxidant status, lipid peroxidation, cytotoxicity, activities of serum enzymes, and hematological parameters. The expression of both proapoptotic and anti-apoptotic genes, and the level of DNA damage were also compared between the control group and MMAE (100 mg/kg b.wt)-treated group.

Estimation of survival time and weight change
The deaths, if any, of the tumor bearing mice were recorded daily and survival time was determined for all the experimental groups. The tumor response following The % increase in median life span (IMLS) and % increase in average life span (IALS) were also calculated using the standard formulae (Gupta et al., 2000). T/C value, which is the MST of the treated group of animals (T) divided by that of control group (C), was also computed. The T/C ratio is given as a percentage and a compound is considered active if it shows T/C value ≥ 120% (National Cancer Institute Protocols). Animals from all the experimental groups were also monitored for alteration in body weight every 3 days up to 18 days posttumor transplantation.

Processing of liver and tumor cell for biochemical assays
After 7 consecutive days of treatment with MMAE or DOX, each animal was euthanized through an overdose of ketamine followed by immediate excision of liver (Al-Batran et al., 2013) that were placed in pre-coded blinded petri -dish. 5% (w/v) tissue homogenate was prepared with ice cold buffer (5 mM EDTA, 0.15 M NaCl, pH 7.4) in a glass homogenizer followed by centrifugation for 30 min at 13,000 rpm at 4 °C. Then, the supernatants were immediately used for biochemical assays. The tumor cells were aspirated in an aseptic condition and washed with NH 4 Cl and 1X PBS twice. The tumor cells were pelleted, sonicated (PCi Analytics) and homogenized with ice cold buffer to produce 5% (w/v) homogenate. Cell homogenates were then centrifuged for 30 min at 10,000 rpm at 4 °C and the supernatants obtained were immediately used for the estimation of antioxidant status and lipid peroxidation.

Antioxidant assays
Protein contents were measured by the standard method (Lowry et al., 1951) using BSA as the standard. Glutathione (GSH) levels were measured by its reaction with DTNB in Ellman's reaction (Moron et al., 1979) to give a compound that absorbs light at 412 nm. GSH concentration was then calculated using the standard graph and represented in μmol/mg protein. Glutathione-s-transferase (GST) was measured using the standard method (Beutler, 1984). GST activity was calculated as follows: GST activity = (OD of test-OD of blank/9.6 × vol. of test sample) × 1000; where 9.6 is the molar extinction coefficient for GST. Superoxide dismutase (SOD) activity was determined by NBT reduction method (Fried, 1975). The enzyme activity was expressed in unit (1 unit = 50% inhibition of NBT reduction/mg protein).

Lipid peroxidation (LPO) assay
The level of lipid peroxidation (LPO) was assessed by the method of Beuege and Aust (1978). Malondialdehyde (MDA) is one of the toxic products formed from the oxidation of fatty acids such as phospholipids and has served as a convenient index for the assessment of the levels of lipid peroxidation reaction. MDA derived from LPO reacts with TBA to give a red fluorescent adduct absorbing at 535 nm. The concentration of MDA in the sample was then calculated using the extinction coefficient of 1.56 × 10 6 M −1 cm −1 .

Effect of MMAE on cell toxicity and hematological parameters
The cytotoxic effect of MMAE was examined by studying tumor cell volume and the percentage of non-viable cell count using trypan blue dye exclusion test, in a hemocytometer. Red blood cell count (RBC), white blood cell count (WBC) and hemoglobin content were measured using standard protocols from the blood obtained by heart puncturing (D' Amour et al., 1965).

Assessment of DNA damage using Comet assay
The alkaline single cell gel electrophoresis (Comet assay) was performed according to the standard method (Singh et al., 1988) with minor modifications. The tumor cells from both control and treatment groups were aspirated in an aseptic condition and washed with NH 4 Cl and 1X PBS twice. Briefly, 2 × 10 4 tumor cells were suspended in 75 µL of 0.5% low-melting point agarose prepared in 1X PBS and spread onto a frosted slide precoated with 1% normal-melting point agarose. Then, the slides were immersed in freshly prepared lysis buffer (10 mM Trizma base, 100 mM Na 2 EDTA, 2.5 M NaCl, 1% Triton X-100 and 10% DMSO, pH 10) for 2 h. After lysis, slides were kept on a horizontal electrophoresis tank filled with freshly prepared alkaline electrophoresis buffer (1 mM Na 2 EDTA, 300 mM NaOH, pH13) for 20 min that will allow unwinding of DNA. Electrophoresis was then carried out at 24 V and 300 mA for 30 min. Then, neutralization was done by washing with buffer (0.4 M Tris-HCl, pH 7.5) for 5 min. After neutralization, slides were washed with distilled water and then stained with ethidium bromide (EtBr) solution (2 μg/mL) for 5 min. Two slides were prepared for each animal and 100 randomly selected cells from each slide were examined using fluorescence microscope (Thermo Fisher Scientific, EVOS R Fluorescence Imaging, AMEP-4615) with a magnification of 200×. Image capture and analysis were performed with Image J software.

qRT-PCR analysis of pro-apoptotic and anti-apoptotic gene expression
The tumor cells from both control and treatment groups were aspirated in an aseptic condition and washed with NH 4 Cl and 1X PBS twice. The cells were pelleted and total RNA was extracted using Tri reagent (BR Biochem, Life Science Pvt. Ltd, R1022). Extracted RNA was quantified using Nanodrop Spectrophotometer (Nanodrop One C, Thermo Fisher Scientific) and RQ1 DNase kit (Promega, M198A, Madison, WI, USA) was used to remove the genomic contamination. cDNA was synthesized from 2 μg of total RNA using first-strand cDNA synthesis kit (Thermoscientific, K1621; Lithuania, Europe). Gene-specific primers (  (Renthlei et al., 2018). Afterward, melting curves were generated to confirm a single uniform peak. GAPDH gene was used as a reference gene for determining the relative expression levels of specific target genes. Each sample was run in duplicate along with non-template and negative RT controls. The relative expression of genes was determined using ΔΔCt method (Livak & Schmittgen, 2001).

Statistical analysis
All data were expressed as mean ± standard error of mean of pooled results obtained from three independent experiments. One-way ANOVA followed by Tukey's test was performed to test significant variations on survival time, change in body weight, antioxidants status, lipid peroxidation, tumor volume, cytotoxicity, hematological parameters, and activities of serum enzymes. Significance variation in relative gene expression and DNA damage were calculated using Student's t test between control and treatment groups. SPSS ver.16.0 software (SPSS Inc, Chicago, Illinois, USA) and Graph Pad Prism ver. 6.0 were used for statistical and graphical analyses. A p value of less than 0.05 was considered statistically significant.

Acute toxicity test
In order to assess the acute toxicity of MMAE, four doses were chosen for the determination of LD 50 starting from 0% mortality to 100% mortality. Thus, four doses were given intraperitoneally to 4 groups of 10 mice each. The dose corresponding to probit 5, i.e., 50%, was found to be 0.182 (log LD 50 ) with LD 50 of 1.52 ± 0.117 g/kg b.wt.

Effects of MMAE on survival time and weight change
To understand the anticancer activity of MMAE on DLA bearing mice, the tumor bearing mice were treated with different doses of MMAE (  Fig. 1A. The weights of the animals in all the groups were recorded every 3rd day starting from the day of tumor transplant in order to determine the change in body weight. Due to the proliferation of the tumor cells, the untreated DLA bearing mice exhibited continuous weight gain until their survival. The treatment of DLA mice with 100 mg/kg MMAE after 72 h of tumor transplantation arrested the weight gain indicating inhibition of tumor cell proliferation and growth (Fig. 1B).

Effect of MMAE on antioxidants/oxidant status
The level and activities of antioxidants and oxidants in DLA bearing mice were measured in the tumor cells and liver after MMAE treatment. Treatment of DLA mice with MMAE results in increased antioxidant levels and activities both in tumor cells and liver when compared with the untreated control. MMAE treatment significantly increased glutathione content in a dose-dependent manner up to 100 mg/kg b.wt when compared to the control group ( Fig. 2A). To determine the effect of MMAE on antioxidant enzymes, the activities of GST and SOD were assessed. In response to MMAE treatment, the antioxidant enzyme activities were significantly increased when compared with the control (Fig. 2B, C). To investigate whether MMAE treatment affects intracellular oxidant level, the level of lipid peroxidation (LPO) was assessed to indicate the level Table 2 Effect of MMAE and DOX treatment on DLA bearing mice on the tumor response assessment based on MST, AST, % IMLS, % IALS and T/C ratio DLA Control: DLA bearing mice without treatment; DLA + MMAE 50, DLA + MMAE 100, DLA + MMAE 150 : DLA bearing mice treated with aqueous extract of M. macrophylla at the dose of 50, 100 and 150 mg/kg, respectively. DOX 0.5 : DLA bearing mice treated with doxorubicin (0.5 mg/kg). The results were expressed as percent (%) ± SEM, n = 10 **p ≤ 0.01; ***p ≤ 0.001between the control and treatment groups Different letters indicates significant variation between different treatment groups ⋆ T/C ratio > 120  of oxidative stress. Consistent to the increased antioxidant enzyme activities, oxidative stress was reduced significantly as evidenced from both the liver and tumor cells after treatment of DLA mice with MMAE. The decrease in LPO level was found to correspond to MMAE treatment in a dosedependent manner up to 100 mg/kg b.wt (Fig. 2D).

Effect of MMAE on tumor volume and cell toxicity
Tumor volume and cell viability were assessed in order to determine the effect of MMAE on tumor load and cytotoxicity. Treatment of DLA mice with MMAE caused a significant reduction in the tumor volume in a dose-dependent manner when compared with the DLA control group ( Table 3). The reduction in tumor volume with MMAE treatment was comparable to the reduction in tumor volume when DLA mice were treated with DOX. Similarly, percentage of non-viable cells has been shown to increase significantly in both MMAE-and DOX-treated DLA mice suggesting the cytotoxic effect of MMAE (Table 3).

Effect of MMAE on hematological and serum biochemical parameters
The DLA bearing mice was shown to have reduced RBC and hemoglobin levels as compared to the normal animals. Interestingly, the decrease in RBC and hemoglobin levels were reversed significantly by MMAE and DOX treatment when compared with the DLA control mice (Table 3). The level of reversal of RBC and hemoglobin contents by MMAE treatment is comparable to the reversal by treatment with the standard DOX treatment. Elevated levels of WBC were observed in the DLA bearing mice as compared to the normal control animals. However, with MMAE and DOX treatment, the WBC levels were significantly reduced to the level close to the normal control group (Table 3). Serum biochemical parameters that include the enzymatic activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine (CRE) level were found to be significantly increased in the DLA control mice as compared to the normal control group. Treatment with 100 and 150 mg/kg b.wt MMAE and DOX (0.5 mg/ kg) were shown to significantly lower the activities of ALT, AST and CRE close to that of the normal control animals ( Table 3).

Induction of DNA strand breaks by MMAE
The alkaline Comet assay was used to assess the level of DNA damage, both double stranded and single stranded, in DLA bearing mice after 7 consecutive days of treatment with MMAE (100 mg/kg). We found that MMAE induced DNA damage in ascites tumor cells which was indicated by significant increased tail length and olive moment in MMAE-treated group when compared to untreated control (Fig. 3).

Effect of MMAE on the expression of p53, Bax, Apaf 1, Bcl-2 and Bcl-X L
The mRNA expression of both pro-apoptotic and antiapoptotic genes were also investigated in DLA bearing mice using qPCR. We found that MMAE induced upregulation of pro-apoptotic genes including p53, Bax and Apaf1 by 4.12-fold, 26.57-fold and 4.51-fold, respectively, and down-regulation of Bcl-2 and Bcl-X L by 2.81-fold and 2.24-fold, respectively, when compared to untreated control (Fig. 4). The relative mRNA expression levels of proapoptotic genes (Bax, p53 and Apaf1) and anti-apoptotic genes (Bcl-2 and Bcl-X L ) in control and MMAE (100 mg/ kg)-treated DLA bearing mice are given in Fig. 4.

Discussion
In order to increase the efficacy of cancer treatment, studies have recently been focused on drugs that have been used in traditional medicine (Singh et al., 2016). Study of the medicinal property of M. macrophylla has mostly been targeted on its antioxidant (Lalremruati et al., 2019), anti-microbial , thrombolytic  and anti-diabetic (Bhandari et al., 2020) activities with no report on its anticancer activity. Therefore, in this study, we investigate the anticancer activities of the aqueous extract of M. macrophylla using DLA bearing mice as our model. Following the standard method of drug administration, DLA mice were treated with MMAE intraperitoneally. The dose used in this study was carefully selected after performing the acute toxicity that gave us the approximate LD 50 which was found to be 1.52 ± 0.117 g/kg b.wt. In all the subsequent analyses, MMAE dose between 50 and 150 mg/kg b.wt was used which are all below the LD 50 .
Several plants of the genus Mussaenda have been known for exhibiting anticancer activities. Mussaenin A, a compound isolated from M. glabrata has been shown to induce apoptosis in Hep G2 via up-regulation of proapoptotic genes (Bax, Bak and Bad) and down-regulation of anti-apoptotic genes (Bcl-2 and Cox-2) (Lipin & Darsan, 2017). Sanshiside D, an iridoid glycoside isolated   Page 11 of 14 Lalremruati et al. The Journal of Basic and Applied Zoology (2022) 83:6 Fleming, 2016). M. roxburghii (Chowdury et al., 2015) and M. luteola (Shylaja & Sathiavelu, 2017) have also been shown to possessed anticancer activities. Interestingly, similar to the other members of the Mussaenda genus, M. macrophylla shows anticancer potential as evidenced from our current study. Our results indicate that MMAE increased the life span of DLA bearing mice as shown by the increase in MST, AST, % IMLS and % IALS (Table 2). Increase in life span of animals is an important criterion for the efficacy of an anticancer agent (Gupta et al., 2004). The body weight of DLA bearing mice generally increases due to the increased cell proliferation of the cancer cells. The potential of MMAE as an anticancer agent was also shown by its ability to suppress the weight gain in DLA bearing mice possibly by inhibiting proliferation of cancer cells in vivo. Cytotoxicity of plant extract is another important feature for consideration as anticancer agent. MMAE treatment was found to exhibit cytotoxicity against ascitic tumors as the treatment reduced tumor volume and increased the percentage of non-viable cells (Table 3). Therefore, our findings suggest that the chemoprotective effects of MMAE could be linked to its role in inhibiting cancer cell proliferation, reduction in tumor load and cytotoxicity which led to increase in life span of the cancer bearing animals. Majority of cancer cells are marked with alteration in hematological parameters (Dongre et al., 2008;Thavamani et al., 2014) as is observed in the case of DLA bearing mice. The hematological changes include reduced levels of RBCs and hemoglobin content accompanied by increased levels of WBC count. Treatment of DLA bearing mice with MMAE showed a pronounced effect in restoring the levels of RBC, hemoglobin and WBC close to that of the normal control levels (Table 3). Inflammation of liver is another feature generally observed in ascitic tumors which could be assessed by determining the levels and activities of key liver enzymes such as AST, ALT, and CRE. In the DLA bearing mice, all these enzymes were highly elevated which was however significantly lowered with MMAE treatment (Table 3). Taking together, MMAE could influence and restore the altered hematological and biochemical parameters in ascitic tumors. The aspect that MMAE could reinstate the altered biochemical and hematological profiles of the DLA bearing mice suggests a promising aspect of M. macrophyllaas an anticancer agent.
The balance between oxidants and antioxidants in the cell of many cancer types remains key to disease progression or improvement. This is accounted to the known role of accumulation of intracellular ROS and its association with cancer progression. Many cancer cells are shown to have higher levels of ROS as compared to the normal cells (Tafani et al., 2016). Therefore, to counterbalance the increased ROS levels in the cancer cell, the cells antioxidant mechanisms have to be efficient. Unfortunately, in many cancers, the activities of the antioxidant system are overwhelmed by the high ROS levels. Therefore, many cancer treatments require external agent to assist the cellular antioxidants. Since high ROS (O 2 ·− , H 2 O 2 and · OH) levels have close association with tumor initiation, angiogenesis, cell invasion, metastasis and chemoresistance in different cancer models (Galadari et al., 2017) use of antioxidants or agents that enhance antioxidant system may provide an opportunity to reduce intracellular ROS-mediated tumorigenesis and cancer progression. In fact, natural products including plant extract have demonstrated antioxidant efficacy such as sesamol, curcumin, ascorbic acid and vitamin E for cancer treatment both in vitro and in vivo (Galadari et al., 2017). Furthermore, a large variety of antioxidants, either alone or in combination with conventional anticancer agents, have been carried out clinically for their use toward anticancer therapeutics in different types of cancer including Acute lymphoblastic leukemia (Al-Tonbary et al., 2009), breast cancer (Zhang et al., 2012) and ovarian cancer (Ma et al., 2014). Consistently, in the present study, augmentation of GSH level and activities of GST and SOD, and decreased lipid peroxidation as evidenced by the significant decrease in MDA levels after MMAE treatment clearly demonstrate its antioxidant nature which may be responsible for its anticancer activity in DLA bearing mice. Over-expression of antioxidant enzymes such as SOD1, SOD2, SOD3,GPx3 and Prx6 has been reported to induce cell death, decrease survival time and suppression of metastasis in various cancer cells (Galadari et al., 2017). Plants such as Hypericum hookerianum (Dongre et al., 2008), Aegle marmelos (Chockalingam et al., 2012), Cyathula prostrate (Mayakrishnan et al., 2014) and Cocculus hirsutus (Thavamani et al., 2014) have been reported to possess anticancer activities in DLA bearing mice via elevation of antioxidant defense system and reduction in lipid peroxidation. Injection of SOD has also been reported to significantly inhibit the peroxidation, metastatic tumor growth and extended the survival period of mice inoculated with B16-BL6 cells (Hyoudou et al., 2008).
DNA damage and induction of apoptosis in response to anticancer agents are another important factor in anticancer therapy. In our study, we observe that MMAE treatment induces significant DNA damage in the ascites tumor cells (Fig. 3). Several plant-derived anticancer drugs have been reported to show similar effects in various cancer types including Dalton's lymphoma (Fattahi et al., 2013;Madunić et al., 2018). The Bcl-2 (B-cell lymphoma/leukemia-2) family of both pro-and antiapoptotic proteins, through their interactions, plays Page 12 of 14 Lalremruati et al. The Journal of Basic and Applied Zoology (2022) 83:6 central roles in regulation of diverse cell death mechanisms including apoptosis (Reed, 2008). Alterations in the expression of these genes contribute to the pathogenesis and progression of cancers, thus providing targets for anticancer drug discovery. Altered expression of anti-apoptotic genes such as Bcl-X L and Bcl-2, and pro-apoptotic genes such as Bid, Bax and Apaf1 have been documented in several human cancers (Sung et al., 2016). Pharmacological inhibition of anti-apoptotic gene expression in cancer has emerged as major strategies for inducing apoptosis and ultimately causing tumor regression (Fesik, 2005). In order to assess the effect of MMAE in inducing apoptosis, the expression levels of apoptotic genes including p53, Bax, and Apaf-1 and anti-apoptotic genes such as Bcl-2 and Bcl-X L were determined. Our result shows up-regulation of pro-apoptotic gene expression while the anti-apoptotic gene expressions were down-regulated (Fig. 4). It is thus plausible that MMAE triggers apoptotic response in DLA mice and offers protective effects in the animals.

Conclusion
Our finding through in vivo cancer model depicts the ability of M. macrophylla to reduce tumor load, prolong the life span of the cancer bearing animals, and activation of apoptotic pathway through DNA damage. It would be interesting to further explore the mechanisms and pathways through which M. macrophylla exerts anticancer effects. Furthermore, it would be important to conduct a bioassay guided fractionation study in order to isolate and characterize the active ingredient that possesses the anticancer activity.