Methanol Extract of Caesalpinia bonducella Induces Apoptosis via Up-Regulation of Bax and Activation of PARP in Ehrlich Ascites Tumor Cells

Nature has endowed plants with the power to cure multitude of diseases. In case of cancer treatment, the search for natural agents with potential antitumor effect has been intensified with time. Here we are reporting analysis of methanol extract of Caesalpinia bonducella (MECB) for its anti-proliferative and pro-apoptotic effect on Ehrlich ascites tumor (EAT) model in-vivo. MECB at 200 mg/kg concentration was responsible for decrease in the total percent of viable EAT cells (51.6%) and ascites volume (65%) in treated mice. MECB increased the rate of apoptosis with more number of cells exhibiting characteristic features such as membrane blebbing, apoptotic body formation and fragmented DNA which was evident by Giemsa and Acridine orange/Ethidium bromide (AO/EB) staining. Alongside, the mice survival time was increased upon MECB treatment. In addition, FACS data suggests that death of MECB treated EAT cells was due to apoptosis and not by necrosis. Furthermore the analysis for molecular mechanism revealed MECB decreased the level of anti-apoptotic Bcl-2 expression while increasing pro-apoptotic Bax level. The immunoblot analysis confirmed the activation PARP (Poly (ADP-ribose) polymerase), a substrate for executioner caspase-3, which brings about subsequent DNA fragmentation in apoptotic process. These results confirm the antiproliferative and pro-apoptotic activity of MECB, which may further translated into therapeutic drug development to combat cancer. Citation: Shivaprakash P, Balaji KS, Lakshmi GM, Chandrashekara KT, Jayarama S (2016) Methanol Extract of Caesalpinia bonducella Induces Apoptosis via Up-Regulation of Bax and Activation of PARP in Ehrlich Ascites Tumor Cells. Med Aromat Plants (Los Angel) 5: 273. doi: 10.4172/2167-0412.1000273


Introduction
Cancer is a multifactorial disease having both genetic factors and environmental risk factors together contributing for the development of disease. Cancer has become second leading cause of death worldwide with higher incidence and mortality rates observed each year, especially in low and middle income countries. As a fact, one in seven deaths globally is attributed to cancer [1,2]. The recent global cancer estimates suggest that nearly 14 million people are diagnosed with cancer in a year and it is been predicted that the number of cancer cases are going to increase up to 19 million by 2025, 22 million by 2030 and 24 million by 2035 [3]. As a consequence, the global market for anti-cancer drugs was soared up to the value of $91 billion in 2013 up from the previous $71 billion in 2008 [4]. Chemotherapy is proved to be an effective and reliable treatment option in cancer therapy [5] which makes use of multiple drugs and hormonal agents alone or in combination [6]. Besides being toxic to cancer cells, the synthetic drugs used for chemotherapy also show lethal effect on normal cells due to their nonspecificity, which just act on dividing cells [7]. The action of these anti-cancer drugs manifests serious side-effects such as dry flaky skin, loss of hair, nausea and vomiting, changes in taste and appetite, blood clotting problems and may cause permanent damage to the kidneys, heart, lungs or reproductive system occasionally [8]. Naturally occurring phytochemicals such as vitamins, flavonoids, alkaloids, diterpenoids, and polyphenols from plants possess multitude of pharmacological effects which have been proved to be potent chemopreventive agents [9]. As a result, 74% of all anticancer drugs currently being used are of natural origin [10]. Yet the quest for medicinal plants with the effective anticancer property is intensified with time.
Caesalpinia bonducella of family Fabaceae (Caesalpiniaceae) is a prickly shrub or woody vine which is worldwide in its distribution [11]. It is widely used in ayurveda -The traditional Indian system of medicine for treatment of variety of diseases such as inflammation, leprosy, malaria, hydrocele etc., [12]. The scientific studies have shown that different parts of the plant possess various pharmacological effects such as antitumor [13], anthelmintic, anti-amoebic and anti-estrogenic [14], analgesic, antipyretic [15], antiasthamatic [16], antibacterial, antidiarrhoel, cytotoxic effect [17], Immunomodulatory activity [18].

In-vivo EAT cell maintenance and treatment with MECB
Ehrlich ascites tumor (EAT), the murine mammary adenocarcinoma cells were grown in 8-10 weeks old female Swiss albino mice by i.p. injections (5 × 10 6 cells /mouse). The mice body weight was monitored regularly for the growth of tumor cells in the peritoneal cavity. After six days of inoculation, the mice were divided in to two groups as control and test. The test group mice received three doses of treatment (i.p.) with MECB (200 mg/kg) on every alternative day starting from day 6 till day 12, whereas control mice received no treatment. On day 13, mice were sacrificed by cervical dislocation and EAT cells along with ascites fluid were collected by making small cut at the peritoneal region, fluid was centrifuged at 2500 rpm for 5 min and the ascites volume was noted down. The cells were washed with physiological saline thrice and used for further analysis. The effect of MECB on cell viability was assessed by trypan blue dye exclusion method [10].

Mice survival analysis
The anti-tumor effect of MECB was also measured by means of survivability time. EAT bearing mice were divided in to two groups of control and treated. At least 10 mice were maintained in each group. The test mice received MECB (200 mg/kg) i.p. and control mice were kept untreated. Mice were monitored regularly with the records of their body weight as long as all mice were succumbed to death.

Apoptotic cell morphology assessment
Apoptotic cell morphology was studied by Giemsa stain and Acridine orange/ Ethidium bromide (AO/EB) double staining method. Briefly, EAT cells were collected by sacrificing both control and MECB treated mice after completion of treatment. Cells were washed several times with physiological saline and fixed in methanol:acetic acid (3:1, v/v). Thin smear of cells was made on glass slide and stained with Giemsa, slides were dipped in distilled water to remove excess dye and observed under light microscope. For acridine orange/ Ethidium bromide staining, the cells were treated with 100 µg/mL AO/EB mixture and observed under fluorescence microscope [19].

Analysis of DNA ladder formation
DNA was isolated according to the procedure reported previously [20]. The cells were lysed with SDS (Sodium dodecyl sulfate. 0.5%) at 37°C and followed by treatment with potassium acetate (8M) for 1 h at 4°C. Cell suspension was centrifuged at 7000 rpm for 60 min, the supernatant obtained was subjected for washing with phenol:chloroform:isoamyl alcohol mixture (25:24:1) for thrice. At the final step of washing, the supernatant was treated with 20 µg/mL RNase at 37°C and was followed with DNA precipitation in chilled ethanol. Finally, the DNA pellet was dissolved in T 10 E 1 buffer for quantification and analysis. The DNA was subjected for agarose gel electrophoresis (1.2%) to assess the characteristic ladder pattern [21].

Detection of apoptosis by flow cytometry
Apoptosis induction by MECB in EAT cells was determined by flow cytometry as reported earlier [22]. Cells were assessed for the loss of mitochondrial membrane potential by staining with a fluorescent dye, tetramethyl-rhodamine methyl ester (TMRM). Cells which were positive for TMRM were considered to have intact mitochondria and the cells with lower fluorescence due to the loss of mitochondrial membrane integrity were served as a reliable marker for apoptosis. Control and MECB treated cells were harvested and washed with cold PBS (phosphate-buffered saline), then were stained with 100 nM TMRM (Molecular Probes-Invitrogen) for 15 minutes at 37°C. Finally the cells were washed with cold PBS and analyzed using BD FACS Calibur (BD Biosciences).

RT-PCR for gene expression analysis
Total cellular RNA was extracted from the cells using Trizol reagent (Invitrogen), Gene expression analysis was performed using Invitrogen Super-script III one-step RT-PCR system. 1 μg RNA was subjected for cDNA synthesis using primer sequences for Bcl-2 (Product No.

Western blot analysis
Western blot analysis was performed following the methods reported elsewhere [22]. According to the method, total protein from the cells was isolated by using lysis buffer containing 0.02 M Tris, 2mM EDTA, 1% Triton X-100, 0.02mM phenylmethylsulfonyl fluoride, 0.1mM NaF, 0.01 mg/mL leupeptin, 0.01 mg/mL pepstatin. Protein was quantified by Bradford protein assay and equal quantity of protein was resolved on 12% SDS-polyacrylamide gel electrophoresis. Protein was transferred on to a PVDF (Polyvinylidene difluoride) membrane and incubated with specific primary and secondary antibodies. The protein bands were detected by ECL plus kit (GE Healthcare).

MECB exerts anti-proliferative and cytotoxic effect on EAT cells in-vivo
To ascertain the anti-proliferative effect of MECB, EAT cells were grown in the peritoneal cavity of mice by injecting 5 × 10 6 cells/mouse (i.p.) and treated with test compound after 6 days of transplantation at the log phase of cell growth. Three doses of MECB (200 mg/kg body weight) was given every alternative day starting from 6 th day, untreated mice were used as control. Mice body weight was monitored every day. As shown in Figure 1A there was considerable decrement in mice body weight upon MECB treatment when compared to untreated control group where gradual increase in mice body weight was observed during the treatment period. The increasing body weight of tumor bearing mice is owing to accumulation of ascites fluid and proliferating EAT cells [23]. Hence the effect of MECB on ascites secretion was assessed ( Figure 1B). The significant reduction in the ascites volume in treated mice (65%) in comparison with control mice was observed. The effect of MECB on EAT cell viability and their number was evaluated by trypan blue dye exclusion method. As a result of cytotoxic effect of MECB the great reduction by 51.6% in total number of viable cells was observed in treated mice when compared to control mice ( Figure 1C). Figure 2 illustrates that as a result of rapid proliferation of tumor cells in the peritoneal region, gradual increment in mice body weight was observed after of transplantation with EAT cells (i.p.). This was also evident from the observation of mice morphological appearance ( Figure 2A). In comparison, the control mice gained significant amount of weight throughout the treatment period where as the EAT-bearing mice treated with MECB showed tremendous reduction in their body weight suggesting the anti-proliferative effect of plant. In addition, MECB considerably increased the survival time of EAT bearing mice up to 34 days but all control mice were died after 14 days of transplantation due to increase of ascites burden ( Figure 2B).

MECB induce EAT cell death in-vivo by means of apoptosis
As a part of cancer treatment, it is imperative to induce apoptosis in tumor cells. Hence the pro-apoptotic effect of MECB was analysed by performing staining techniques and DNA fragmentation assay. Figure  3A and 3B depicts Giemsa staining and EB/AO staining respectively. MECB treated EAT cells displayed typical apoptotic morphologies such as cellular shrinkage, chromatin condensation and formation of apoptotic bodies in comparison with intact control cells. Further, the effect of MECB on induction of EAT cell apoptosis was confirmed by resolving DNA from both control and treated cells on agarose gel. MECB induced apoptosis was confirmed from the characteristic DNA ladder pattern which is one of the hallmark of apoptotic cells ( Figure 3C).

Influence of MECB on apoptotic rate of EAT cell
In the intrinsic pathway, DNA damage induces release of cytochrome c from the intermembrane space leading to failure in maintaining the mitochondrial membrane potential and ATP synthesis [24]. At the onset of apoptosis, activated caspase-3 cleaves PARP, which results in the formation of PARP cleavage products, a hallmark of apoptosis [25,26]. In the intrinsic or mitochondrial pathway of apoptosis, caspase activation is closely connected to the permeabilization of the outer mitochondrial membrane [27]. The changes in mitochondria   membrane potential were investigated using TMRM staining. The rate of apoptosis induction by MECB was assessed by FACS analysis. EAT Cells from control and treated mice were stained with 100 nM TMRM for 15 min and pro-apoptotic effect was determined by measuring mitochondrial membrane potential. As illustrated in Figure 4, the rate of EAT cell apoptosis was remarkably higher in MECB (200 mg/kg) treated mice, over 43.7% of total cell population were observed. We found that MECB induced loss of mitochondrial membrane potential (Δψ m ) in EAT cells, which indicated that the mitochondrial apoptotic death-signal pathway plays a critical role in MECB induced apoptosis in EAT cells.

Effect of MECB on Bcl-2, Bax and PARP expression in EAT cells
Bcl-2 and Bax are the important signalling factors of intrinsic apoptotic (mitochondria mediated) process [28] that regulate cytochrome c release, activation of caspases and DNA fragmentation [29]. Bcl-2, the member of Bcl-2 family is an oncoprotein that resist cell death where as Bax, another constituent of the Bcl-2 family acts as pro-apoptotic factor by inducing apoptosis. Studies have shown that the expression of pro-apoptotic proteins such as Bax or Bak is very necessary to induce cellular apoptosis [30]. The ratio of bcl-2 (antiapoptotic) / bax (pro-apoptotic) majorly determines the sensitivity of a cell to death signals [31]. Alteration in the Bcl2 family protein expression has been associated with many cancer types whereas Bcl2 over expression in tumor cells is regarded as a common attribute [32]. Previous reports have shown the effects of medicinal plants in down regulating Bcl-2 protein expression in EAT cells [19,33]. To determine the effect of MECB on Bcl-2 and Bax, total cellular RNA was extracted and subjected for reverse transcription-PCR using specific primers. As it is shown in Figure 5A, MECB reduced anti-apoptotic Bcl-2 expression to great extent in EAT cells while upregulating the expression of proapoptotic protein Bax. Thus MECB is shown to have modulatory effect on apoptosis signalling proteins.
Poly (ADP-ribose) polymerase (PARP) is a DNA repair enzyme and is cleaved by activated effector caspases-3, activated PARP brings about subsequent DNA degradation in apoptosis process [34]. To determine the involvement of MECB in caspase induced DNA fragmentation immunoblot analysis was performed. Figure 5B reveals that MECB induce the cleavage of PARP and confirms the likely activation of executioner caspase-3. The rate of apoptosis induction by MECB was assessed by FACS analysis. Cells from control and treated mice were stained with 100 nM TMRM. As it is illustrated in Figure, the rate of apoptosis was remarkably higher in MECB treated mice (200 mg/kg) in comparison with control mice alongside. Apoptosis was detected by measuring mitochondrial membrane potential.

Figure 5: Effect of MECB on Bcl-2, Bax and PARP expression in EAT cells:
To determine anti-apoptotic (Bcl-2) and pro-apoptotic(Bax) expression level in EAT cells upon MECB treatment, total cellular RNA was extracted from the cells of both control and treated mice and subjected for RT-PCR analysis. (A) As it is illustrated in the picture, MECB considerably decreased the expression level of Bcl-2 while showing positive effect on Bax expression (B) Graph represents the Bcl2/Bax ratio in control and MECB mice. (C) Immunoblot analysis was performed for the detection of cleaved Poly (ADP-ribose) polymerase (PARP) as it is shown, MECB induced PARP activation confirms the caspase mediated apoptosis. β-Actin was used as internal loading control in the experiment.

Conclusion
The present study emphasizes in-vivo cytotoxic effect of methanol extract of C. bonducella leaves on EAT cell model. The experimental results shows that induction of apoptosis by mitochondrial pathway which is mediated by alteration mitochondrial membrane potential and Bax up regulation. The upregulated Bax leads to the caspase-3 activation, which in turn cleaves of PARP. Further the identification bio active components which brings cell apoptosis is essential for a promising source for developing novel therapeutics to combat cancer.