Regulation of apoptosis through bcl-2/bax proteins expression and DNA damage by Zanthoxylum alatum

Abstract

Context: Many of the major chemotherapeutic agents are secondary metabolites found in nature. Zanthoxylum alatum Roxb. (Rutaceae) is traditionally used in the treatment of various diseases.

Objective: The present study evaluates the apoptotic activity of methanol extract of Z. alatum (MEZA) on Ehrlich ascites tumor (EAT) in Swiss albino mice.

Materials and methods: The presence of flavonoids in MEZA was standardized by HPLC. The in vitro cytotoxicity of MEZA was measured by the MTT assay. The in vivo antitumor activity of MEZA (100 and 200 mg/kg b.w., i.p. for 9 days) was also evaluated. On the 10th day, EAT tumor volume, cell viability, and hematological parameters were assayed. Apoptotic morphology was determined by acredine orange/ethedium bromide using fluorescence microscopy. Apoptosis percentage was measured by flow cytometric analysis using annexine-V-FITC. Also, DNA damage and bcl-2/bax were estimated by UV-method and western blot, respectively.

Results and discussion: HPLC analysis revealed presence of three flavonoids, rutin, myricetin, and quercetin. MEZA showed satisfactory cytotoxicity in MTT assay (IC₅₀ = 111.50 µg/ml). The extract significantly (p < 0.01) changed the tumor volume, viable, non-viable cell count, and hematological parameters towards the normal. Apoptotic activity of MEZA was confirmed by acridine orange/ethidium bromide staining, annexin-V-FITC staining, DNA fragmentation, and Bcl-2/Bax ratio.

Conclusion: The study showed that MEZA has antitumor activity which may be due to the presence of flavonoids in the extract.

• Cytotoxicity
• Ehrlich ascites tumor
• flavonoids
• western blot

Introduction

Nature is an excellent and reliable source of new drugs, including anticancer agents. Around 70% of the currently used anticancer chemotherapeutic drugs are derived from natural sources (Patra & Muthuraman, 2013). Among the secondary metabolites, flavonoids, such as tangeretin, nobiletin, hesperidin, and naringenin have gained limelight in recent times with their potential anticancer activities (Batra & Sharma, 2013).

Zanthoxylum alatum Roxb. (Rutaceae) is an evergreen plant of the Himalayan regions in India, commonly known as Tejphal (Hindi) and Timur (Nepal) (Singh & Singh, 2011; Tiwary et al., 2007). The ethnomedicinal importance of its seeds has been well known for a long time in Indian medical system as a stomachic, carminative, disinfectant, and antiseptic; and for the treatment of fever, dyspepsia, cholera, anthelmintic, general debility, and preventing snake bites (Jain et al., 2001; Kalia et al., 1999; Prakash et al., 2012). Nepalese traditionally used the fruit decoction in abdominal pain; bark extract for cholera, diabetes, and asthma. Pickles from the fruits are used by Nepalese for treating colds and coughs, tonsillitis, headache, fever, and high-altitude sickness (Geweli & Awale, 2008).

Scientific bioactivity determination studies have revealed its larvicidal (Tiwary et al., 2007), hepatoprotective, antioxidant (Ranawat et al., 2010), antinociceptive, anti-inflammatory, and antipyretic activities (Guo et al., 2011). Various phytopharmaceuticals, such as berberine, dictamnine, xanthoplanine, armatamid, asarinin, fargesin, α- and β-amyrins and lupeol are present in the plant (Kalia et al., 1999; Nadkarni, 2002). The present study evaluates the apoptotic activity of the methanol extract of Z. alatum leaves through bcl-2/bax pathway and other supportive parameters.

Materials and methods

Animals

Swiss albino mice of about 8 weeks of age with an average body weight of 20–25 g were used for the experiment. Animals maintained under standard environmental conditions (approved by Animal Ethical Committee, Jadavpur University, Kolkata, India), were fed with a standard diet (Hindustan Lever, Mumbai, India) and water ad libitum. The animals were acclimatized to laboratory condition for one week prior to the experiment. The animals were fasted for 16 h before experimentation but allowed free access to water.

Plant collection, authentication and extraction

Zanthoxylum alatum leaves were collected in the month of July 2012, from the hilly region of Gangtok, India. The plant material was authenticated by the Botanical Survey of India, Howrah, West Bengal, India. A voucher specimen (CNH/38/2014/Tech. II/78) has been preserved in our laboratory for future reference. The leaves were cleaned and air dried for a week at 35–40 °C and pulverized in electric grinder. Powdered leaves (1.1 kg) were consecutively extracted by petroleum ether, chloroform, and methanol by using a Soxhlet apparatus. The methanol extract of Z. alatum (MEZA) (10.6%, w/w, yield) was selected for evaluation of apoptotic activity.

High-performance liquid chromatography characterization of flavonoid

The presence of flavonoid compounds in MEZA were determined by Dionex Ultimate 3000 HPLC system (Dionex, Dreieich, Germany), using a reverse phase C-18 column (250 × 4.6 mm, particle size 5 µ) and an UV detector. The samples were prepared by dissolving in HPLC methanol and filtered through cellulose nylon membrane filter (0.45 µm) (Pall Life Sciences, Bangalore, India). The aliquots of the filtrate were eluted with isocratic solvent mixture comprising methanol:acetonitrile:acetic acid:orthophosphoric acid:water (20:10:1:1:20) for flavonoids with a flow rate of 1 ml/min and detected at 352 nm.

Acute toxicity

MEZA (2 g/kg) was administered orally to male Swiss albino mice to evaluate the acute toxicity as per the reported method (Chatterjee et al., 2013).

Transplantation of tumor cell

Ehrlich ascites tumor cells were maintained in our laboratory according to the standard protocol (Haldar et al., 2010).

In vitro cytotoxicity assay

In vitro cytotoxicity of MEZA was determined using previously used standard MTT assay (Nikhil et al., 2014). The experiment was performed in triplicate.

Treatment schedule for assessment of in vivo antitumor potential

Treatment schedule was maintained according to the previously used experiment (Dolai et al., 2012) in our laboratory. Swiss albino mice (20–25 g) were divided into five groups (n = 10).

• Gr. I: normal control mice administered normal saline (5 ml/kg, i.p) daily for 9 days;

• Gr. II: EAT-induced mice received normal saline (5 ml/kg, i.p) daily for 9 days;

• Gr. III: EAT-induced mice treated with MEZA (100 mg/kg, i.p.) daily for 9 days;

• Gr. IV: EAT-induced mice treated with MEZA (200 mg/kg, i.p.) daily for 9 days;

• Gr. V: EAT-induced mice treated with 5-fluorouracil (5-FU) (20 mg/kg i.p) daily for 9 days.

Tumor related parameters

The mice were dissected and the ascitic fluid was collected from the peritoneal cavity. Percentage increase in life span (% ILS), tumor cell (viable/non-viable) count were calculated according to the standard methods (Karmakar et al., 2013).

Hematological parameters

Collected blood was used for the estimation of hemoglobin (Hb), red blood cell (RBC), and white blood cell (WBC) count by standard procedures (Karmakar et al., 2013).

Morphological studies by fluorescence microscopy

Morphological changes of EAT were carried out in three groups of mice (Gr. II–IV) using acridine orange/ethidium bromide staining (Arimura et al., 2003; Verma & Prasad, 2012) followed by an observation under a fluorescence microscope (Leica, Wetzlar, Italy) using a blue filter and photographed.

Western blot analysis

Sample protein (Gr. II–IV) was isolated as per the protocol mentioned by Dua et al. (2015). Samples containing proteins (20 μg) were subjected to SDS-PAGE (12%) and transferred to a nitrocellulose membrane following the standard dry transfer protocol. Membranes were blocked (4 °C; 1 h) using Tris-buffered saline with 0.1% Tween 20 (TBST) containing non-fat dry milk (5%) to prevent non-specific binding with gentle shaking. The membranes were washed with TBST (pH 7.6) for three times (5 min, each) with gentle shaking. Then the membranes were incubated with primary antibodies, namely, anti-Bax (1:1000), anti-Bcl-2 (1:1000), in TBST containing 5% of BSA at 4 °C overnight with gentle shaking. The membranes were washed with TBST (pH 7.6) for three times (5 min, each) with gentle shaking and incubated with appropriate HRP-conjugated secondary antibody (1:3000 dilution) in TBST containing non-fat dry milk (5%) for 1 h at room temperature and finally developed by the HRP substrate 3,3-diaminobenzidine tetrahydrochloride system (Bangalore Genei India Private Limited, Bangalore, India). The western blot analysis and densitometry studies were performed using Image Lab 5.2 software (Bio Rad, Hercules, CA).

Detection of apoptosis by annexin-V-FITC binding assay

The EAT cell lysate (Gr. II-IV) were treated with propidium iodide (PI) and FITC-labelled Annexin V for 30 min at 37 °C. Excess of PI and Annexin V were then washed off; cells were fixed and analyzed by flow cytometry using FACS Calibur (Becton Dickinson, Mountain View, CA) equipped with 488 nm argon laser light source; 515 nm band pass filter for FITC-fluorescence and 623 nm band pass filter for PI-fluorescence using Cell Quest software. A scatter plot of PI-fluorescence versus FITC-fluorescence has been prepared (Pal et al., 2011).

DNA fragmentation analysis of EAT cells

Ehrlich ascites tumor cells (1 × 10⁶) were collected from EAT control and treated groups of mice (Gr. II–IV) and DNA fragmentation in the EAT cell was determined by the method as described by Lin et al. (1997).

Statistical analysis

Statistical analysis was performed using Graph Pad Prism (version 5.0, GraphPad Software Inc., San Diego, CA) Software. All data were expressed as mean ± standard error of mean (S.E.M.). The data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. Results were considered statistically significant when p < 0.05.

Results

Detection of flavonoid

The flavonoid compounds were identified employing reverse phase HPLC analysis and comparing retention time (Rt) and UV spectra with standard flavonoids compounds. HPLC analysis revealed presence of flavonoids (Figure 1), namely rutin (Rt: 3.00), myricetin (Rt: 3.9), and quercetin (Rt: 5.6) in MEZA. In this study, a number of standard flavonoid compounds were used to identify their presence in MEZA. The chromatograms of standard compounds were represented based on the abundance of phytochemicals in MEZA.

Figure 1. HPLC chromatograph of standard flavonoids and MEZA where (1) rutin (Rt: 3.0), (2) myricetin (Rt: 3.9), (3) quercetin (Rt: 5.6).

Detection of acute toxicity

The extract was safe up to the dose of 2 g/kg b.w. p.o. for mice and 100 and 200 mg/kg b.w. doses were used in the present study.

Detection of in vitro cytotoxicity

The IC₅₀ value of in vitro cytotoxicity after 24 h was found to be 111.50 by plotting the graph of concentration versus percentage inhibition (Figure 2).

Figure 2. Cytotoxic effect of MEZA on in vitro EAT cell line. Values are mean ± S.E.M.; where n = 3.

Detection of tumor-related parameters

Antitumor activity of MEZA against EAT-bearing mice was assessed by the parameters, such as tumor volume, %ILS and cell count (viable and non-viable). Administration of MEZA at doses of 100 and 200 mg/kg significantly (p < 0.01) decreased the tumor volume and viable cell count, whereas non-viable cell count was significantly (p < 0.01) higher as compared to EAT control animals (Table 1). Furthermore, the %ILS was increased to 37.90 and 67.61% on administration of MEZA at 100 and 200 mg/kg, respectively (Table 1).

Table 1. Effect of MEZA on tumor volume, viable and non-viable cell count, percentage increase life-span (% ILS) and hematological parameters of normal and EAT-bearing mice.

Parameters	Gr. I	Gr. II	Gr. III	Gr. IV	Gr. V
Tumor volume (ml)	–	2.34 ± 0.14	1.92 ± 0.11*	1.68 ± 0.17*	0.84 ± 0.09*
Viable cell ( × 10^7 cell/ml)	–	7.26 ± 0.47	4.51 ± 0.31*	2.84 ± 0.17*	0.92 ± 0.11*
Non-viable cell ( × 10^7 cell/ml)	–	0.28 ± 0.06	1.14 ± 0.12*	2.96 ± 0.34*	3.52 ± 0.21*
% ILS	–	00	37.90	67.61	96.43
RBC (cell × 10^6/µl)	5.67 ± 0.13	3.54 ± 0.58^a,*	3.91 ± 0.31^b,*	4.86 ± 0.36^b,*	5.23 ± 0.56^b,*
WBC (cell × 10^3/µl)	5.26 ± 0.51	9.59 ± 0.94^a,*	8.02 ± 0.42^b,*	6.71 ± 0.61^b,*	5.92 ± 0.25^b,*
Hemoglobin (g/dl)	11.95 ± 1.05	6.63 ± 0.28^a,*	7.76 ± 1.04^b,*	9.59 ± 0.76^b,*	10.41 ± 0.61^b,*

Values are represented as mean ± S.E.M., where n = 6.

^aEAT control group versus normal group, ^btreated groups versus EAT control group, *p < 0.01.

Detection of hematological parameters

Hematological parameters (RBC, WBC, and Hb) of MEZA treated and untreated tumor-bearing mice were evaluated (Table 1). Treated groups (Gr. III and Gr. IV) showed significant (p < 0.01) increase in the level of both the RBC count and Hb content, while WBC count was reduced when compared with the EAT control group.

Detection of morphological changes

A fluorescence microscopic analysis demonstrated that untreated EAT cells were stained with a uniform green fluorescence (Figure 3A), whereas the MEZA (100 and 200 mg/kg) treated cells stained with greenish-orange fluorescence indicated apoptotic properties, like membrane blebbing and nuclear condensation (Figure 3B–C).

Figure 3. Fluorescence microscopic images of EAT cells from Gr. II (control EAT cells), Gr. III (EAT cells treated with MEZA 100 mg/kg), and Gr. IV (EAT cells treated with MEZA 200 mg/kg) using acridine orange and ethidium bromide. Arrows indicate the formation of apoptotic bodies, condensed nucleus and membrane blebbing as an evidence of MEZA induced apoptosis. Magnification (100×).

Study of western blot analysis

The western blot analysis was performed to observe expression changes of Bcl-2 and Bax proteins during MEZA-induced EAT cell apoptosis. As shown in Figure 4(A), at different time intervals after treatment with MEZA, a decreased expression of Bcl-2 was evident, while that of Bax protein began to increase. Therefore, our data suggest that the ratio of apoptotic antagonist (Bcl-2) and apoptotic agonist (Bax) is also related to MEZA-induced apoptosis (Figure 4B).

Figure 4. Western blot analysis of Control (Gr. II) and treated EAT cell (Gr. III and IV). This blot was of three independent experiments, where β-actin was used as the loading control. Data are represented as means ± S.E.M. of three separate experiments.

Detection of apoptosis percentage

To understand the nature of cell death, we utilized double labeling techniques using Annexin-V-FITC/PI to distinguish between apoptotic and necrotic cells. Our flowcytometric data revealed that, in comparison with control untreated EAT cells (Figure 5A), MEZA at the doses of 100 and 200 mg/kg increase the apoptosis level by13.4 and 17.6%, respectively (Figure 5B–C).

Figure 5. Flow cytometry analysis of MEZA-treated EAT cells. Cells were labeled with annexin V-FITC and PI. (A) Untreated cells, (B) cells treated with MEZA (100 mg/kg), and (C) cells treated with MEZA (200 mg/kg).

Detection of DNA fragmentation

The percentage of DNA fragmentation in the EAT cell of the experimental mice is represented in Figure 6. MEZA (100 and 200 mg/kg) treatment significantly increased (p < 0.05) the extent of DNA fragmentation as compared with EAT control. This indicates that the apoptotic role of MEZA in EAT cells ruled out the possibility of it being necrotic to EAT cells.

Figure 6. Effect of MEZA (Gr. III and IV) on DNA fragmentation of EAT cell in experimental mice. Values are expressed as mean ± S.E.M., for five animals in each group. ^aValues differ significantly from EAT control (*p < 0.05).

Discussion

Flavonoids belong to a very vast group of plant secondary metabolites with variable phenolic structures and are found in almost every part of the plant (Nijeveldt et al., 2001). Many epidemiological studies have been conducted to prove the protective effect of flavonoids against cancer. It has been reported that compounds (rutin, myricetin, and quercetin) from different plant sources have efficacy against different cancer (Batra & Sharma, 2013).

The EAT cells were initially described as a spontaneous murine mammary rapidly growing adenocarcinoma with a very aggressive behavior and can proliferate in almost all strains of mice (Dolai et al., 2012). The EAT implantation induces a local inflammatory reaction, with increasing vascular permeability, which results in an intense edema formation, cellular migration, and a progressive ascites fluid accumulation (Bala et al., 2010).

The results show that administration of MEZA at doses 100 and 200 mg/kg significantly reduce the ascitic volume, tumor cell count (viable), and inhibit the proliferation of EAT cell growth in vivo. These suggest that MEZA may have direct relationship with tumor cells as these tumor cells take up the anticancer drug by absorption within the peritoneal cavity, which lyse the cells by direct cytotoxic mechanism (Kennedy et al., 2001). The increase in life span of tumor-bearing mice by decreasing the nutritional fluid volume and arresting the tumor growth is a positive result and supports the antitumor effect of MEZA (Dolai et al., 2012).

Myelosupression and anemia are the major adverse effects of cancer chemotherapy (Haldar et al., 2010). Administration of MEZA restored the hemoglobin content, RBC, and WBC count towards normal, thereby exhibiting its protective role on a hemopoietic system.

Acridine orange is taken up by both viable and non-viable cells. Green fluorescence is emitted when the dye is intercalated double-stranded nucleic acid (DNA) or red fluorescence when bound to single-stranded nucleic acid (RNA). Ethidium bromide is taken up only by non-viable cells and red fluorescence is emitted when intercalated into DNA (Curcic et al., 2012). Different characteristics of EAT cells like fluorescence, blebbing type, and nuclear condensation help to distinguish the apoptotic, necrotic, and normal cells.

Members of the Bcl-2 family of proteins interact to regulate apoptosis through the mitochondrial pathway (Ramos, 2007). The significant up-regulation of proapoptotic factor Bax promotes apoptosis. The up-regulation of Bax might restrict the entry of cells into S-phase and can promote nuclear DNA fragmentation (Widlak, 2000). The result shows that treatment with MEZA decreased Bcl-2 and simultaneously increased Bax levels, resulting in the decrease of Bcl-2/Bax ratio (Chatterjee et al., 2013). The balance between the pro- and anti-apoptotic proteins thus shifted towards apoptosis. However, the presence of high levels of Bax by itself is not sufficient to induce apoptosis without an additional stimulus. Therefore, involvement of other apoptogenic pathways in EAT apoptosis cannot be ruled out.

In the early stages of apoptosis, the cell membrane is still intact and impermeable to DNA binding dye PI. But Annexin V binds specifically with phosphotidylserine, is translocated to the extra-cellular leaflet of the membrane. In contrast, during necrosis, because the cell membrane is ruptured, these cells take up both the fluorochromes. The MEZA-induced cell death observed in this study can occur by two distinct modes – apoptosis and necrosis, which can be distinguished by morphological and biochemical features. Annexin-V-FITC/PI staining of MEZA-treated EAT cells resulted in an increase in Annexin⁺/PI⁻ and Annexin⁺/PI⁺ cells compared to the control (Annexin^-/PI^-), indicating apoptosis as a possible mode of cell death (Sharma et al., 2012).

Conclusion

This is a significant study investigating the effects of flavonoid-enriched Z. alatum extract in EAT cells and exploring different mechanisms underlying their cytotoxic potential. Flavonoids have the potential of modulating many biological events in cancer, such as apoptosis, vascularization, cell differentiation, cell proliferation, etc. EAT cells exposed to flavonoid-enriched MEZA showed a decrease in cell viability and the mode of MEZA-induced cell death was found to be apoptosis. Based on our data, we can assume that MEZA induced apoptosis probably by activation of both intrinsic and extrinsic pathways due to the presence of flavonoids. These results support the ethnopharmacological use of Z. alatum as an anticancer agent. Future investigations will focus on cell cycle and to determine the role of p53, p21, TNF alpha, and extrinsic apoptotic pathways.

Acknowledgements

The authors thank the Jadavpur University and Indian Institute of Chemical Biology (IICB), Kolkata, India, for their technical support.

Declaration of interest

The authors declare that they have no conflict of interest. They also acknowledge the Department of Science and Technology (DST), Govt. of India, for the INSPIRE fellowship [IP12048] sanction no. DST/INSPIRE Fellowship/2012/423 to the first author (IK).