Plant-derived Tetranortriterpenoid, Methyl Angolensate Activates Apoptosis and Prevents Ehrlich Ascites Carcinoma Induced Tumorigenesis in Mice

Background: Cancer is a leading health problem throughout the world. For decades, natural plant products have been playing promising roles as anticancer agents. Objective: The present study aims to investigate the chemotherapeutic potential of methyl angolensate (MA), purified from Soymida febrifuga in mice bearing carcinoma and examines the molecular basis for its anticancer actions. Study Design: The inhibitory effects of MA treatment on the survival of mice bearing Carcinoma and adverse side effects of MA treatment in mice were analyzed. Methods: Tumor volume, life span, histopathology, immunohistochemical (IHC) analysis, estimation of liver enzyme, alkaline phosphatase and metabolites, creatinine and urea. Results: Oral administration of MA in mice with Ehrlich Ascites Carcinoma showed significant Original Research Article Chiruvella et al.; BJMMR, 12(11): 1-10, 2016; Article no.BJMMR.21952 2 inhibition of tumor growth compared to untreated mice. We observed a significant increase in the life span (~4-fold) of tumor bearing animals following treatment with MA. MA affected tumor cell proliferation by activating intrinsic pathway of apoptosis without imparting any side effect on normal cells. MA treatment in mice showed no major side effects. Conclusion: MA treatment showed significant inhibition of tumor growth by inducing apoptosis as well increased life span of mice, with no adverse side effects to normal cells. Altogether, the present in vivo study provides new insights of MA serving as a cancer chemotherapeutic agent.


INTRODUCTION
Cancer continues to be a leading health problem throughout the world, despite momentous research efforts towards development of therapeutics.
Previously, utilizing cell based assays, we reported that MA exhibited time-and dosedependent cytotoxicity to human T-cell leukemia, chronic myelogenous leukemia, Burkitt's lymphoma and breast cancer cells [13][14][15][16]. The observed cytotoxicity in normal lymphoblastoid cell lines was minimal suggesting that its mode of action was specific to cancer cells. Besides, treatment of MA led to activation of NHEJ machinery in Burkitt's lymphoma cells suggesting activation of DNA repair pathway [16]. Strikingly, MA treatment also resulted in phosphorylation of signaling molecules such as ERK1/2, JNK and MEK1/2 in cancer cells implying their role in apoptotic signal transduction. Although the action of MA against various types of cancer cell lines suggested its potential as an anticancer agent, its effects in primary tumor cells or tumors in vivo remained unexplored.
Here, we show that MA impedes tumor progression in mice similar to a positive control, SCR7, a small molecule inhibitor that possess anticancer properties. Histopathological and immunohistochemistry studies demonstrate that MA treatment affects only tumor cell proliferation, sparing normal cells. Further, our data indicates that MA activates intrinsic pathway of apoptosis. Finally, we show that treatment with MA results in significant increase in life span of mice with no adverse effects.

Methyl Angolensate (MA)
MA used in the present study was dissolved in 0.5% methyl cellulose and used for animal experiments.

Ethical Statement
Mice were maintained as per the principles and guidelines of the ethical committee for animal care at Indian Institute of Science, which was in accordance with Indian National Law on animal care and use. Tumor volume was measured every alternate day during the entire life span and was calculated using the formula V = 0.5 x a x b 2 , where 'a' and 'b' indicates the major and minor diameters, respectively [17][18][19][20]. At the end of 25 th and 45 th day, one animal from each group was sacrificed and organs of interest were collected from untreated control, tumor control and MA treated animals and further processed for histological evaluation.

Determination of Effect of MA on Survival of Tumor Bearing Mice
The percentage increase in life span was calculated by using the formula ((T − C)/C) × 100, where 'T' indicates the number of days the treated animals survived and 'C' indicates the number of days the control tumor animals survived [17][18][19][20][21]. Animals were maintained till their natural death. However, few animals which were high in tumor burden were sacrificed before their natural death.

Determination of the Side Effects of Administration of MA in Mice
Serum was collected from blood of control (n=4) and MA administered (n=4; 30 mg/kg b. wt., 9 doses, every alternate day). Body weights of mice from both the groups were recorded 20 days after treatment. Liver and kidney function tests were performed as described, to check the toxicity induced by MA treatment [17,22]. Levels of alkaline phosphatase activity served as an index of liver functionality. Kidney function was determined by checking urea and creatinine levels in the serum. Blood collected from mice in both the groups was used for obtaining mean values of total red blood (RBC) and white blood cells (WBC) counts using a haemocytometer.
Values are presented as mean ± SEM for the control and MA administered mice.

Histopathological Evaluation Immunohistochemical (IHC) Analysis
The tissues and organs of interest (tumor and liver) collected from the animals were processed as per standard protocol and embedded in paraffin. Microtome sectioning was done at 3-5 μm in a rotary microtome (Leica Biosystems, Germany) and stained with hematoxylin and eosin [17,18,22,23]. Each section was evaluated using light microscopy and images were captured (Carl Zeiss, Germany).

Immunohistochemical (IHC) Analysis
Immunostaining of tumor and MA treated tissue was performed on formalin fixed, paraffin embedded tissues and sectioned at 3-5 μm thickness as described [18,22]. Rabbit polyclonal antibodies Ki67, BID and p53BP1 (Santa Cruz Biotechnology) and biotinylated anti-rabbit IgG were used. Multiple images from control and treated groups were quantified using ImageJ.

Statistical Analysis
Values were expressed as mean ± SEM for control and experimental samples and statistical analysis was performed by one-way ANOVA followed by Turkey-Kramer Multiple Comparison test using GraphPad prism 5.1. Values were considered statistically significant, if the p-value was less than 0.05.

MA Prevents Tumor Progression in Mice
In order to evaluate the potential of MA as an anticancer agent, mice harboring EAC induced tumor were utilized. EAC is a murine breast adenocarcinoma extensively used for analyzing effects of small molecule inhibitors on tumor progression. A pilot study was performed using EAC induced Swiss albino mice for determining the dose of MA needed for tumor regression (Fig.  1B). The tumor induced mice were treated with two doses of MA (10 or 30 mg/kg b.wt) or an equivalent volume of vehicle control. Interestingly, we found that, while tumor volume increased exponentially in the untreated group, in both the treated groups the tumor progression was reduced (Fig. 1B). The tumor regression was pronounced in the group treated with 30 mg/kg b.wt. MA, while it was minimal when the lower dose was used (Fig. 1B). Importantly, the mice showed no observable difference in appearance or body weight (data not shown) implying that MA did not adversely affect its health.
Based on the above results, therapeutic potential of MA was extensively assayed in three independent batches of experiments (in each experiment, 10 each untreated normal control, untreated tumor control and tumor mice treated with MA) at a dose of 30 mg/kg b.wt. (Fig. 1C). Following the development of tumor, 12 th day post EAC injection, the mice were orally fed with MA on every alternate day (9 doses). Fig. 1C showed remarkable reduction in tumor size upon treatment with MA as compared to untreated animals. Fig. 1D shows average tumor volume in untreated animals was 14.78, 13.84, 18.94, 23.89 and 38.76 cm 3 on 10 th , 20 th , 30 th , 40 th and 50 th day respectively, whereas it was only 6.56, 5.22, 6.16, 1.57 and 0.0 cm 3 respectively, upon MA treatment (Fig. 1C). We found that 83% (25/30) of the mice with tumor were dead by 50 th day of tumor development when left untreated. Interestingly, 70% (21/30) of the mice survived following treatment with MA up to at least 300 days (Fig. 1D).
SCR7 is an inhibitor of nonhomologous DNA end joining (NHEJ), and shown to possess antitumor activity in mice [18,[24][25][26]. Therefore, we have used SCR7 as a positive control for tumor regression. Upon treatment with SCR7 in mice bearing EAC induced tumors we found significant tumor regression in tumor volume, while the growth was exponential in the untreated group (Fig. 1E).

MA Treatment Significantly Increased the Survival of Tumor Bearing Mice
We observed lifespan was significantly increased in MA treated tumor mice compared to that of untreated tumor animals (Fig. 1D). While control animals survived maximum of only 68 days after tumor development, 50% of the MA treated mice survived upto >200 days and 25% of the mice survived upto 300 days indicating significant increase in life span (Fig. 1D). Therefore, our results suggest that MA treatment inhibited tumor progression and improved the survival of mice, significantly.
Next, we performed histopathological examination of tumor and liver tissues of MA treated mice on 25 th and 45 th days, for analyzing cellular level changes and toxicity caused by MA. Hematoxylin-eosin stained sections of 25 th and 45 th day untreated tumor showed cellular damage, angiogenesis and tumor cell proliferation ( Fig. 2A (b, e), B (b, e)) as compared to normal untreated mice thigh tissue ( Fig. 2A (a,  d), B (a,d)). However, tumor tissues showed very high tumor cell proliferation and complete destruction of cellular architecture. Interestingly, tumor tissue from MA treated mice showed only mild cell architecture damage, while proliferation of tumour cells was reduced ( Fig. 2A  (c, f), B (c, f)). When similar tissue sections were examined after 45 days of tumor development, the effect was very prominent following MA treatment and it could be seen that the tissues were restored to normal cellular morphology (Fig. 2B (c, f)).
Histopathological sections of liver from normal mouse showed proper arrangement of hepatocytes with no infiltration (Fig. 2C (a, d), D (a, d)), while liver from tumor bearing mouse after completion of 25 days of treatment showed infiltration of hepatocytes, cellular damage and dilation of central veins ( Fig. 2C (b, e), D (b, e)). In contrast, liver from MA treated mouse showed dilated sinusoids with minimum infiltration of hepatocytes and nearly normal rearrangement of hepatic cell chords (Fig. 2C (c, f)). However, the effect was more pronounced when tissues were analysed following 45 days of MA treatment (Fig. 2D (c, f)). Thus, our results showed that MA treatment significantly reduced the tumor burden.

MA Abrogates Tumor Progression with no Side Effects
Next we evaluated the side effects of MA treatment for serum levels were analysed for liver enzyme such as Alkaline phosphatase (ALP), and metabolites like creatinine and urea. Results showed no significant difference in the serum levels of ALP, creatinine and urea between untreated control and MA treated mice groups suggesting that there were no adverse effects of MA on liver and kidney functions (Fig. 3A). Further, we did not observe any effect of MA on the total RBC and WBC counts suggesting that it did not affect hematopoiesis. Analysis for toxicity on 0 th day after oral administration of MA showed that there was no significant change in the body weight among the control and treated groups (Fig. 3B). Thus, our data indicates that MA treatment does not result in any apparent side effects in normal mice.

MA Induces Cell Death through Activating Apoptosis
Previously studies on cancer cell lines suggested that MA induced apoptosis by activating   9 doses) and untreated mice, were tested for various toxicological parameters after 20 days of treatment. A. Blood was collected and serum levels were checked for liver and kidney enzymes such as alkaline phosphatase (ALP), creatinine and urea. Variation in the counts of RBC and WBC were also compared following MA treatment. B. Body weight (g) was measured to assess the effect of MA following 20 days of oral administration. In all the cases, error bars indicate mean ± standard error mean mitochondrial pathways. In order to investigate whether the same holds true in tumor tissues, immunohistochemical analysis was performed using antibodies against protein markers of cell proliferation and apoptosis in histological sections of mouse tumor tissues following MA treatment (30 mg/ kg, 9 doses, 25 th day) (Fig. 4). We observed efficient Ki67 nuclear staining in untreated tumor sections ( Fig. 4A (a, g)). while number of Ki67 positive cells were substantially less in MA treated tumors, although there was no significant difference in staining pattern between control and treated groups ( Fig. 4A (b, h)). Besides we observed that the expression of proapoptotic protein, BID was significantly high following MA treatment compared to untreated tumor tissues (Fig. 4A). This indicates the activation of intrinsic pathway of apoptosis in MA treated tumor tissues, which is consistent with our earlier studies that shows that MA induces intrinsic pathway of apoptosis [13,15,16]. Further, we also tested the expression of DNA repair protein, p53 binding protein, p53BP1. The expression of p53BP1 was significantly high in MA treated tissues compared to that in tumor tissues (Fig. 4B). Hence, our results suggest that MA treatment resulted in the abrogation of tumor cell proliferation by activating the apoptotic pathway in mice induced with carcinoma.

DISCUSSION
Compounds purified from medicinal plants have been considered as valuable sources for development of anticancer drugs. Differential effects of these natural products on tumor cells could be due to their difference in the efficacy to induce apoptotic pathways and this also might play a significant role in their chemopreventive potential.
Among various plant derived molecules, limonoids are triterpenoids, which are biologically important and possess tumor preventive properties as shown by several in vitro and in vivo studies. Our previous studies, utilizing various cell line based assays, identified that MA possesses anticancer properties [13,15,16]. However, this is the first study to test the anticancer properties of MA in primary tumor tissues using an in vivo model system.
Here, we have uncovered that MA has the potential to cause tumor regression in mice bearing breast adenocarcinoma. Further, this study, in conjunction with previous ex vivo studies show that MA activates intrinsic pathway of apoptosis to inhibit tumor growth [13,15,16].   [13,16]. We also tested the therapeutic potential of MA (30 mg/kg b. wt., 9 doses) in the Dalton's lymphoma mouse model. Our results showed no reduction of tumor in MA treated animals (data not shown). These results suggest that MA was effective against tumor induced by breast adenocarcinoma cells, while it was insensitive to tumors generated by Dalton lymphoma cells.
Apoptosis is one of the important mechanisms, which determines the efficacy of anticancer drugs. Our previous findings showed that, upon treatment with MA, intrinsic pathway of apoptosis leading to cell death was activated in both leukemia and lymphoma cells. During apoptosis, proapoptotic proteins promote the release of mitochondrial Cytochrome C into cytoplasm, while antiapoptotic proteins block the Cytochrome C release by preserving the integrity of mitochondrial membrane. Previously, it has been shown that MA treatment resulted in increased BAD and decreased BCL2 expressions leading to an altered ratio of antiapoptotic/proapoptotic proteins [13]. The increase in the BAD/BCL2 ratio resulted in disruption of mitochondrial membrane potential and Cytochrome C release, which in turn activated Caspase 9 and Caspase 3 culminating in cell death, indicating that MA displayed activation of the intrinsic pathway of apoptosis [13].
In the present study, we measured expression of one of the proapoptotic proteins, (BID) in tumor tissues. An increase in BID expression in MA treated tumor tissues as compared to that in untreated tissues suggest that MA increased the levels of BID, causing its translocation from the cytosol into mitochondria. Thus, our findings in conjunction with previous studies suggested the activation of mitochondrial pathway of apoptosis following treatment with MA in tumor tissues. Similar activation of the intrinsic pathway has also reported in the case of other terpenoids [30][31][32]. DNA damage is a critical event preceding cellular apoptosis. Accumulation of DNA double strand breaks leads to activation of apoptosis in cancer cells. The observed elevated expression of 53BP1 in tumor tissues was also consistent with this. Overall, DNA strand breakage following MA treatment in tumor cells supports the notion of activation of apoptosis in cancer cells and recapitulates DNA fragmentation induced by MA in cancer cell lines during apoptosis.

CONCLUSIONS
Taken together, our data suggests that MA abrogated tumor progression blocking cell proliferation by inducing intrinsic pathway of apoptosis in EAC induced mice as well increased life span of mice, with no adverse side effects to normal cells. Therefore, our current study in conjunction with previous reports reveals the potential of plant-derived MA as a potential cancer chemotherapeutic agent.

CONSENT
It is not applicable.