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In Vitro Cytotoxic Activity against Human Lung Carcinoma Cell Lines (A549): Identification and Quantification of Quercetin, A Major Constituent of Artocarpus altilis by Targeting Related Genes of Apoptosis and Cell Cycle

Tara K Jalal*

Department of Biomedical Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia (IIUM), Pahang, Malaysia

*Corresponding Author:
Tara K Jalal
Department of Biomedical Science, Kulliyyah of Allied Health Sciences
International Islamic University Malaysia (IIUM), Jalan Sultan Ahmad Shah
Bandar Indera Mahkota, 25200 Kuantan, Pahang
Malaysia
Tel: 006095739110
E-mail: [email protected]

Received date: March 28, 2017; Accepted date: April 05, 2017; Published date: April 10, 2017

Citation: Jalal TK (2017) In Vitro Cytotoxic Activity against Human Lung Carcinoma Cell Lines (A549): Identification and Quantification of Quercetin, A Major Constituent of Artocarpus altilis by Targeting Related Genes of Apoptosis and Cell Cycle. Nat Prod Chem Res 5: 261. doi: 10.4172/2329-6836.1000261

Copyright: © 2017 Jalal TK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Nine phenolic compounds were identified and quantified in A. altilia fruit. One of the main compounds was quercetin, which is the major class of flavonoids which has been identified and quantified in pulp part of A. altilis fruit of methanol extracts. The aim of this study was to evaluate the in vitro cytotoxic assay study of the flavonols: quercetin, on A549 (human lung carcinoma cells). Furthermore, the pulp of the fruit was down-regulated the expression of anti-apoptosis gene BCL-2 and up-regulated the expression of pro-apoptosis gene BAX. CASPASE-3 was also activated by the fruit, which started a CASPASE-3-depended mitochondrial pathway to induce apoptosis. It was also found that the pulp of methanol extracts was arrested the cells in G0/G1 and G2/M phases. As the results, the pulp was the most active in terms of all tests, due to high amount of quercetin in pulp part 78% of total flavonoids. Taken together, these findings suggested that A. altilis induces apoptosis in a mitochondrial-dependent pathway by releasing and up-regulating CYTOCHROME C expression and regulates the expression of downstream apoptotic components, including BCL-2 and BAX. Pulp part of the methanol extracts can be a potent and promising medicine as anticancer agent.

Keywords

Quercetin; Apoptosis; Cell cycle; CASPASE-3

Introduction

Cancer is an irregular growth of cells caused by genetic alterations that lead to the deregulation of cell proliferation and cell death. Sridharan et al. reported that cancer is a genetic disorder of the cancerous cells [1]. Cancer is initiated by external causes (tobacco, infectious organisms, chemicals, and radiation) and internal causes (inherited mutations, hormones, immune conditions, and mutations originating from metabolism).

In 2000, approximately 10 million people were diagnosed with cancer when the global population was approximately 6 billion which 5.3 million patients were men and 4.7 million were women. In 2014, approximately 176,000 of the estimated 585,720 cancer deaths were caused by tobacco consumption. According to the World Cancer Research Today, 24.6 million people are living with cancer, and 6.7 million of them are dying of cancer every year. Parkin et al. reported that a gradually increasing ratio of older people in the world will result in approximately 50% increase in new cancer cases over the next 20 years [2]. More men than women suffer from the cancer of the lung, stomach, throat, and bladder. In wealthier countries, prostate, breast, and colon cancers are more common than in poor countries [3]. External factors such as tobacco consumption, exposure to chemicals and radiation, infectious organisms, and internal factors such as inherited mutations, hormones, and immune status can cause cancer. These dangerous causes may act together or in sequence to initiate or promote carcinogenesis. The American Cancer Society reported that more than 175,000 cancer deaths were caused by the consumption of tobacco in year 2005 [4]. A study in 2015 reported that approximately 14.1 million incidences and 8.2 million mortality related to cancer occurred in 2012. Globally, cancer (lung and breast) has been in top frequently diagnosed cancer for both man and woman respectively [5]. Thus, noticing the global pattern, this study chooses lung and breast cancer to understand the effects of porcupine bezoar in using both cancer cells as a model. In the lung cancer study, in 2015 incidence reported as the second leading in male (14%) and female (13%). In 2016, though the percentage rate of incidence retains, but the cases had increased by 2310 for man and 880 cases for woman [5,6]. It seems the lung cancer mortality rate was highest for male and female for both years. In 2015 the mortality rate of lung was 28% (total case of 312 150) for male and 26% (total case of 277 280) for female. The rate was decreased in 2016 for male by 1% (total case of 314 290) while for female remains 26% (total case of 281400) [5,6]. It has been discovered that flavonoids have many beneficial actions on body cells by enhancing the activity of many enzyme systems, effective in inflammation, arteriosclerosis, bleeding, allergy and swellings. It is also known to be associated with reduced risk of certain types of cancers. However, the major problem associated with the use of quercetin, is the very low bioavailability biological activities of flavonoids such as anti-allergic, anti-toxic, anti-microbial, anticataract and anti-cancer.

Materials and Methods

Materials

The plant sample was collected from Taman Pertanian Sultan Haji Ahmad Shah, Kuantan, Pahang. The parts of A. altilis fruits were prepared for extraction by washing off all dirt and soil residues then, peel off all the skin. Cut the pulp to even parts. Then the dried pulp of A. altilis were ground to a fine powder, and then stored in a cold room at 4°C until further analyses. The ground plant materials were extracted using a Soxhlet apparatus according to Akbar et al. [7]. A sample mass of 250 g pulp of Artocarpus altilis were extracted by filling the absorbent cellulose thimble and placing it in the thimble chamber of the Soxhlet apparatus. Three solvent systems (hexane, dichloromethane and methanol) were used for 12-18 h in sequence in order of increasing polarity to obtain three different types of extracts - i.e., hexane extract of pulp; DCM extract of pulp and methanol extract of pulp. In the current study, only the methanol extract of the pulp going to be used. The solvent was removed by rotary evaporation at 60°C in vacuo.

Cell line and cell culture

Human lung carcinoma (A549) was obtained from American Type Culture Collection (ATCC). The preparation of complete growth media (CGM) used were Dulbecco`s modified Eagle medium (DMEM), L-glutamine supplemented with 10% fatel bovine serum (FBS) and 100 U/mL penicillin, 100 mg/mL streptomycin. Media were aliquoted into 50 mL centrifuge tube, kept in 4°C and pre-warmed to 37°C prior to use. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C and 95% air. All handling processes of the cells were performed under strictly aseptic techniques inside the class II biosafety cabinet and performed as rapidly as possible to minimize contamination.

Determination of inhibitory concentration 50% (IC50)

A stock solution of test sample (100 mg/mL) was prepared by dissolving tested sample of pulp of methanol extracts in DMSO (0.1 g/1000 μL). To minimise the DMSO effect in the sample, 1 mg from the stock solution (100 mg/mL) was prepared by aspirating 10 μL from the stock and dissolved into 990 μL of complete culture medium. It was then gently shaken using vortex shaker. This 1 mg sample is the working solution and is kept at 4°C until use. It is preferable to prepare these fresh at the time of the experiment. Setting approximately 5 × 104 cells per well, in the 6-well plates of A549 cell lines. After 24 h, the partial monolayer was formed; the supernatant was cleared off, washed 1-2 times with PBS (Gibco BRL., UK). Serial dilutions of 1 mg solution were prepared in the culture medium in 6-well plates ranged between 12.5-200 μg. Table 1 shows the working solution concentrations of methanol pulp extract solution which was freshly prepared prior to use for further assays.

Volume that added to the well from stock solution (1 mg/mL) Volume of CGM/well/µL Final concentrations (µg/mL)
400 1600 200
200 1800 100
100 1900 50
50 1950 25
25 1975 12.5

Table 1: List of concentrations of pulp extracts used in this study.

Test sample at the appropriate concentrations was added to individual wells as mentioned, while only adding complete DMEM growth medium for the control (untreated, no extracts added). As for screening, a series of concentration range from higher to lower concentration of the extract of pulp of A. altilis were carried out in triplicate. The plates were then incubated at 37°C in 5% CO2 incubator for 72 h [8]. After 72 h incubation, the medium was aspirated, the cells harvested and the IC50 concentration was determined using TBEA method briefly, using a haemocytometer, which is a device used to count cells with a special type of microscope slide consisting of two chambers, by uploading 10 μL of diluted cell suspension (10 μL) is a mix of trypan blue and cell suspension (1:1) Strober [9]. The experiment was repeated in triplicate and analysed using GraphPad Prism 6.01 software.

Identification and quantification of phenolic compounds using UHPLC-MS/MS

Analyses of the phytochemicals components of A. altilis of pulp part was performed on an AB Sciex 3200QTrap liquid chromatographytandem mass spectrometry (LCMS/MS) (AB Sciex, Toronto, Canada) coupled to Perkin Elmer Flexar FX15 ultra-high-performance liquid chromatography (UHPLC) system (Massachusetts, USA), operated by AB Sciex analyst software. Reference standards were also used for comparison. The retention times of the extract with those of the reference standards were compared for further confirmation and identification. The external standard method was used for the quantification of the individual phenolic compounds. Contents of 9 phenolic compounds such as quercetin, rutin, ascorbic acid, p-coumaric acid, ferulic acid, gallic acid, 4-hydroxybenzoic acid, protocatechuic acid and sinapic acid, were calculated with the regression equations from the standard curves. The standard calibration curve of each of the standard was used to quantify the number of phenolic compounds present in the various crude extracts.

Flow cytometric analysis of apoptosis and cell cycle

In order to determine the level of apoptosis in cells, Guava Nexin reagent staining, a pre-made cocktail containing Annexin V-PE and 7-AAD in buffer. All adhering and floated cells were harvested and pipetted 100 μL of each sample into appropriate well using 96-wells, then 100 μL Guava Nexin reagent was added to each well, incubated for 20 minutes at room temperature in the dark. Sample was acquired on Guava flow cytometry system. The results were analysed using guava Incyte software, version 2.7, which 2000 cells were collected for the analysis. While for cell cycle after cells were harvested, and transferred to sterile centrifuge tube, then discard the supernatant and washed with PBS centrifuge, the supernatant was removed and the pellet resuspended with 70% cold ethanol (400 μL) and kept at -20°C for 1-2 h. The cells were washed with PBS, centrifuge and discard the supernatant. Then 200 μL of PI kit (containing 1 mg/ml RNAse) was added to the cell pellets and incubated in the dark for 30 minutes at room temperature. The cells were then analysed by Guava flow cytometry, 5000 cells were collected for analysis using guava Incyte software, version 2.7.

Quantitative Real-Time PCR

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis

RNA extraction and cDNA preparation: The process of exerting RNA and converting to cDNA were carried out according to manufacturer’s instructions. Total RNA was isolated from selected treated and untreated cell lines using innuPREP RNA Mini Kit (Konrad-Zuse-Strasse, Germany) after the appropriate drug incubations for 72 h of both control and treated cells. Total RNA 100 ng was used for cDNA synthesis in a final volume of 20 μL by using reverse transcription system SensiFAST™ bioline kit, preforming the cDNA step by setting in the thermal cycler. The thermal cycler was fixed up at the following program: 25°C for 10 min (primer annealing); for the reverse transcription, the program set up was at 42°C for 15 min; for inactivation, it was set at 85°C for 5 min; and finally set at 4°C to keep the product cold. Using the comparative Cq method, mRNA expression levels of target genes were normalized to the expression of glyceraldehyde phosphate dehydrogenase (GAPDH) and β-actin (ACTB) as endo genes.

Statistical analysis

Data of replicates were analysed using a one way ANOVA analysis and were expressed as means ± standard error (SE) mean of the triplicates. Statistical differences between the reference and the sample groups were evaluated by ANOVA (one way) using LSD (least significant differences) test p< 0.05 using SPSS 20.0 software. And used for calculating IC50 Graph pad prism version 6.01.

Results

Determination of pulp of methanol extracts that effectively inhibits 50% of A549 Cells

Figure 1 shows the graph obtained by the log value of the agonist concentration (in M) versus the percentage of cells viability as compared to the untreated cells to determine the IC50 value of the methanol extracts on A549 cells, following incubation for 72 h. The IC50 of the pulp of the fruit extract was found to be 23.10 ± 0.71 μg/mL.

natural-products-chemistry-methanol-extracts

Figure 1: Concentration-dependent effect of methanol extracts of pulp part on the percentage of cells viability of A549 cells. Data presents the mean ± standard error mean (SEM) of mean of triplicate analyses of two independent experiments (n=6).

Identification of various phenolic compounds in A. altilis of pulp extract using UHPLC-Ms/Ms

The identification of phenolic compounds was classified into flavonoids and phenolic acids. Two of the compounds were identified as flavonoids and seven as phenolic acids (Table 2).

Mass spectra
Compound identity Standard RT (min) UV (nm) M−H−(m/z) MS2 (m/z)−
Quercetin 4.20 360 301 151
Ferulic acid 3.55 232 193 178
4-Hydroxybenzoic acid 2.90 280 137 93
Sinapic acid 3.57 323 223 193
Protocatechuic acid 2.53 219 153 109
Rutin 3.38 255,352 609 301
p-Coumaric acid 3.34 223,309 163 119
Gallic acid 2.06 271 169 125
Ascorbic acid 1.31 265 175 87

Table 2: Identification of phenolic compounds in A. altilis.

Flavonoids: Flavonoids have attracted widespread attention recently because of their broad range of beneficial effects on human health. The best-described characteristics of almost every group of flavonoids are their ability to remove free radicals and inhibit other oxidation reactions [10]. Compounds belonging to various flavonoid classes (quercetin and rutin) were detected in A. altilis samples of pulp analysed. Peak 1, (Figure 2) presented spectral characteristics of quercetin [11]. Quercetin’s spectral characteristics are: UV−vis spectrum at 360 nm, M− H− at m/z 301 and fragment ion at m/z 151 (Table 2).

natural-products-chemistry-phenolic-compounds

Figure 2: The chromatograms of phenolic compounds presented in pulp part of A. altilis methanol fruit extracts.

The standard chromatograph of quercetin is shown in Figure 2. Lepley et al. and Sergediene et al. reported that numerous phenolic compounds have been shown to exhibit antiproliferative and cytotoxic effects on several tumour cells, and presented toxic effects that specifically target cancer cells rather than normal cells [12,13].

Quercetin is a powerful antioxidant, and the most abundant in dietary flavonols. It is found in abundance in fruits, vegetables, beverages and berries [14]. This compound was said to have very powerful chemopreventive and chemotherapeutic (anticancer) potential [15]. Kamaraj et al. reported quercetin’s anticancer properties against benzo (a) pyrene- induced lung carcinogenesis in mice which was attributed to its free radical scavenging activity [16].

Peak 2 was identified as rutin based on its M− H− at m/z 609 and fragment ion at m/z 301 which was also previously reported by Alakolanga et al. [17]. They also provided the reference chromatogram of rutin (Figure 2). Rutin is found in citrus fruits [18], and has been associated with potential health benefits. Additionally, aside from their antioxidant properties, the flavonoids are powerful agents against chronic diseases such as cardiovascular diseases, atherosclerosis, and malignancies [19-21].

Phenolic acids: Phenolic acids (p-coumaric acid, ferulic acid, gallic acid, 4-hydroxybenzoic acid, protocatechuic acid, sinapic acid and ascorbic acid) were the most identified in A. altilis extracts.

Peak 3 in with M H at m/z 193 and fragment ion at m/z 178 (Table 2) were identified as ferulic acid according to the mass spectral library and reference standard (Figure 2). In addition, the mass spectrum was in agreement with those reported by Gómez-Romero et al. [22]. Paiva et al. reported that ferulic acid-rich dates has been shown to have antioxidant, anti-microbial, anti-inflammatory, hepatoprotective, neuroprotective, anticarcinogenic, anti-diabetic and anti-cholesterolemic properties [23].

The MS Peak 4 values at m/z 137 (M H) and UV-vis spectrum 280 nm unambiguously identified it as 4-hydroxybenzoic acid. This is based on a similar compound reported in Taraxacum Formosa num, a Chinese medicinal herb grown in Taiwan [24].

The retention time and fragment ion (m/z 93) were also identical to those of 4-hydroxybenzoic acid standard. In a study by Seidel et al., [25], 4-hydroxybenzoic acid did not only arrest cell cycle progression but also triggered apoptotic cell death in cancer development.

Peak 5 is shown in Figure 2 was characterized as sinapic acid based on UV-vis spectrum at 323 nm and M− H− at m/z 223 [26]. In addition, the fragment ion at m/z 193 was in agreement with a reference standard. This compound is one of the biologically active components of many fruits, vegetables, cereal grains, medicinal plants, and spices. Sinapic acid had been reported for its various biological activities such as anti-inflammatory [27], antibacterial [28] and antidiabetic activities. It has been shown to possess anticancer effects in different cancer cell lines such as antiproliferative, antiapoptotic properties and is also able to arrest the cell cycle [29].

Peak 6 was identified as a protocatechuic acid. This compound was previously detected in abundance in edible fruits and vegetables and is thus one of the antioxidative components of normal human diet [30]. Protocatechuic acid isolated from the Chinese herb Salvia miltiorrhiza, played a crucial role against inflammatory cytokines of atherosclerosis. Hu et al. reported that protocatechuic acid has the ability of inhibiting both the invasion and metastatic potential of malignant carcinoma cells [31].

Peak 7 is shown in Figure 2 showed a mass spectral characteristic of p-coumaric acid having M− H− at m/z 163 and fragment ion at m/z 119. Some identified phenolic acids including p-coumaric, ferulic, and sinapinic acids have been earlier shown to inhibit the growth of some cancer cell lines [32-34]. The peaks were identified as p-coumaric acid and was confirmed by comparing these values with that of the standard p-coumaric acid.

Peak 8 show M− H− at m/z 169 and fragment ion at m/z 125. These were identified as gallic acid [26]. Gentisic acid, p-coumaric acid, ferulic acid and gallic acid elicited a significant increase in the activities of several antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) in rats [35].

By comparing the UV-vis spectrum at 265 nm, M− H− at m/z 175 and fragment ion at m/z 87 with those reported in literature [36], ascorbic acid peak 9, was identified. It is a naturally occurring organic compound with a powerful antioxidant properties and immune response activation. It is involved in wound healing and osteogenesis. It can be found in fresh vegetables and citrus fruits [37].

Quantification of phenolic compounds in A. altilis

Calibration curves were generated from the reference standards after the identification of phenolic compounds in the extracts. Linear regression equation obtained from the calibration curve of each standard was used for the quantification of phenolic compounds of A. altilis extracts. Concentrations of individual and total phenolic compounds (expressed in mg per kg dry weight), linear regression equations and regression coefficients (R2) are presented in Table 3.

Compound Concentrations pulp Regression equations R2
Flavonoids  
Quercetin 43.20 Y=4.04e+006x +4.38e+005 0.9987
Rutin 12.20 Y=8.64e+005x+4.73e+004 0.9997
Total 55.40  
Phenolic acids  
Ferulic acid 3.29 Y=2.09e+006x+2.32e+005 0.9990
4-Hydroxybenzoic acid 1.56 Y=7.81e+006x+2.19e+006 0.9995
p-Coumaric acid 8.33 Y=5.27e+006x+1.01e+006 0.9997
Protocatechuic acid 11.40 Y=5.69e+006x+1.12e+006 0.9996
Sinapic acid 0.28 Y=9.86e+005x+6.38e+004 0.9997
Gallic acid 1.24 Y=3.53e+006 x+4.86e+005 0.9987
Ascorbic acid 0.17 Y=5.03e+005 x+6.64e+004 0.9984
Total 26.27  

Table 3: Concentrations (mg/kg dry weight), regression equations and regression coefficients (R2) of phenolic compounds in A. altilisextract.

Flavonoids varied from 12.20 to 43.20 mg/kg dry weight for pulp. The range for phenolic acids in the present study were from 0.17 to 11.40 mg/kg dry weight for pulp (Table 3).

Quercetin was the most concentrated and abundant flavonoid in pulp amounting to 78.00% followed by MW and ML at 40.96% and 32.47% of the total flavonoids concentration, respectively. Rutin was (20.02%) of the total flavonoids concentration.

Protocatechuic acid was the most concentrated phenolic acid present in plup part which accounted for 43.36% of the total phenolic acids.

Followed by p-coumaric acid in pulp was 31.70% of the total phenolic acids concentration. Ferulic acid was 4.09%.

Both the sinapic acid and ascorbic acid were the least components found in pulp part (1.06% and 0.41%). Gallic acid was at 4.00%. While the percentage of 4-hydroxybenzoic was 5.93%. These results indicate that the composition of phenolic compounds can vary between different parts of the same fruit. To date, this is the first report that investigates crude extracts of A. altilis fruit showing important bioactive compounds and the analysis of pulp of methanol extract of A. altilis fruit using LCMS UHPLC.

A. altilis induced apoptosis studied by nexin staining

To determine whether cytotoxic effect of A. altilis was due to apoptotic induction, methanol extracts using IC50 were induced early and late apoptosis at 72 h (Figure 3). The percentages of both early and late apoptosis were induced by pulp part of the fruit on A549 cell line for early apoptosis 22.98 ± 0.10 and 32.26 ± 0.26% compared to untreated cells 3.22 ± 0.11% at p< 0.05. Figure 3a demonstrates the raw data of the dot plot of the cell populations distributed through the quadrant marker.

natural-products-chemistry-Apoptosis-induced

Figure 3: Apoptosis induced by A. altilis fruit extracts on A549 cells. Cells were treated with a concentration of IC50 for 72 h. Data presents as the mean ± standard error mean (S.E.M.) of mean of triplicate analyses. Different letters indicate significant differences between treated cells upon IC50 of methanol extracts and relative respective control of each group (untreated) at p<0.05.

natural-products-chemistry-Effects-on-apoptosis

Figure 3a: Effects on apoptosis induction of A549 upon treatment of IC50 of A.altilis at 72 h. A (untreated) cells and B (treated) cells.

Effects of A. altilis extracts on cell cycle activity arrest on A549 cells upon treatment with IC50 at 72 h

Figure 4 show the cell cycle arrest of A549 cells at 72 h. At 24 h the treated cells showed some decrease in cells population at G0/G1 and S phases 38.01 ± 0.56 and 8.75 ± 0.24% compared to untreated cells 48.21 ± 0.13 and 16.50 ± 0.52% (Figure 4a), respectively, at p< 0.05. While at G2/M phase the cells showed increment mediated by pulp 49.43 ± 0.72%, compared to untreated cells 33.72 ± 0.26%.

natural-products-chemistry-Cell-cycle-arrested

Figure 4: Cell cycle arrested by A. altilis fruit extracts upon treatment of IC50 on A549 cells for 72 h. Data presents as the mean ± standard error mean (S.E.M.) of mean of triplicate analyses. Different letters indicate significant differences between treated cells upon IC50 of methanol extracts and relative respective control of each group (untreated) at p<0.05.

natural-products-chemistry-Effects-of-cell-cycle

Figure 4a: Effects of cell cycle arrest of A549 cells upon treatment of IC50 of A. altilis at 72 h. A (untreated) cells and B (treated) cells.

Induction of apoptotic and cell cycle gene expressions in A549 Cells by A. altilis methanol extract

Critical exposure of the methanol extract of A. altilis to A549 cells moulded different expression profiles for apoptotic and pro-apoptotic genes (CASPASE-3, 8, 9, BAX, BCL-2, FAS-L, CYTOCHROME C and cell cycle gene (p21, CYCLIN-A1, CYCLIN-B1 and CDK1) mRNA levels. As shown in Figure 5, the mRNA level of CASPASE-3 and 8 were induced at highest level than other genes and also as compared to the control as consider is one P< 0.05. Figure 5 shows the expression level of methanol extracts of pulp induced apoptotic signals via intrinsic pathway on A549 cell lines by upregulating FAS-L and CASPASES-8 with 20.44 ± 1.17 and 36.23 ± 3.41.

natural-products-chemistry-PCR-analyses

Figure 5: Real-time PCR analyses of selected genes that were modulated in A549 cells by pulp methanol extract treatment according to cDNA. The effects of fruit part on the expression levels of apoptosis genes FAS-L, CASPASE-3, CASPASE-8, CASPASE-9, BAX, BID, BCL2, CYCS1, and cell cycle genes p21, CYCLIN A1, CYCLIN B1, CDK1 and CDK2 are shown. Total RNA samples were isolated from A549 cells treated with IC50 μg/mL of pulp methanol extract for the 72 h. The modulations of mRNA expression levels were expressed as fold change based on calculation using GAPDH and ACTB as HKGs, assigning the ratio in untreated cells as 1. Data are presented the mean ± standard error mean (S.E.M.) of mean of triplicate analyses of NRQs. *Indicate significantly different from control at p<0.05.

As can be observed from the results, a high fold ratio was obtained by pulp giving opportunity for mitochondrial pathway via upregulation and release of CYCS1 of 29.70 ± 2.80. This in turn upregulated CASPASE-9 to 21.59 ± 0.60. Whereas the relationship of the CASPASE-3 and P21 is very evident, as can be observed from the results that when the ratio expression level of CASPASE-3 is high the p21 also high 27.81 ± 2.65 and 36.23 ± 3.41 CASPASE-3 and p21, respectively.

As observed from the results the fold changes of the genes’ expression upregulated due the exposure to the treatment of IC50 for 72 h. The pulp arrested the A549 cells at G2/M by downregulating the CYCLIN B1 and its kinase CDK1. p21 is able to influence and block this binding process at G2/M checkpoint by interfering with the CDK1/ CYCLIN B1, (Figure 5). Figure 6 presents the proposed schematic representation of the death signals of A549 cells after the treatment. It followed the extrinsic pathway via activation of the death receptor FAS-L over CASPASE-8 then cleaves BID into tBID to initialize the mitochondrial pathway, activating the BAX then release the CYTOCHROME C activation of CASPASE-9 and finally CASPASE-3 [6].

natural-products-chemistry-schematic-model

Figure 6: Proposed schematic model for apoptosis induction and cell cycle regulation in A549 cells affected by pulp extract for 72 h.

Discussion

The impact of new and novel agents from potential bioactive plants or their extracts for disease treatment and prevention is still massive, even though the research contribution by synthetic chemistry and chemists, as a method of drug discovery and drug manufacture is intense [38]. Many studies have focused on a new strategy in the fight against cancer due to the chemoprotective posibilities of plants having anticarcinogenic properties. Phytochemicals, including specific phenolic compounds from plants, seems to play a significant role in suppressing all three stages of tumour formation, i.e., initiation, promotion, and progression [39]. Natural products show promise for cancer chemoprevention using medicinal plants. These medicinal plants provide non-cytotoxic nutrients or pharmacological agents to enhance the physiological mechanisms that protect the organism against mutant clones of malignant cells [40]. Earlier studies on Artocarpus plants have shown various responses to extracts. Wang et al. reported that five geranyl dihydrochalcones from the ethyl acetate extract of A. altilis leaves had cytotoxic effects on some human cancer cell lines [41], such as human lung adenocarcinoma (SPC-A-1 cells), human colon carcinoma (SW-480 cells), and human hepatocellular carcinoma (SMMC-7721 cells). Plant extracts and the bioactive compounds present in them, which are responsible for anticancer activity have to be screened for valuable information and classification into various classes of anticancer agent. Natural phytochemicals can be categorised into carotenoids, phenolics, alkaloids, nitrogen-containing compounds and organosulfur compounds.

These phytochemicals are responsible for various pharmacological actions like antioxidant, and anticancer properties [42]. Yang et al. and Lee et al. reported that quercetin have been known to induce strong antiproliferative properties of a wide range of cancers such as prostate, cervical, lung, skin, stomach and colon cancer cells in three leukemic cell lines (CEM, K562 and Nalm6) [43,44], two breast cancer cell lines (T47D and EAC) compared to other tested polyphenols [45-48]. As reported earlier (Table 2 and Figure 2) pulp contained high percentage of quercetin and is considered the major compound 78% of total flavonoids. Thus, pulp promotes more activity towards selected cell lines.

Results show pulp contain rich flavonoids mainly quercetin. The presence of a hydroxyl group for the methoxy group at the meta position of the phenyl ring significantly enhanced the proliferative activity [49]. The second major compound that was identified and quantified in pulp was protocatechuic acid (phenolic acids) which had been previously shown to inhibit the growth of some cancer cell lines such as human breast cancer MCF-7 cells, lung cancer A549 cells, HepG2 cells, cervix HeLa cells, and prostate cancer LNCaP cells [50]. Senawong et al. reported that p-coumaric acid (a naturally occurring bioactive phenolic acids in fruits), the third major compound that was identified and quantified in pulp 31.70% of the total phenolic acids concentration was found to induce cytotoxicity and increased anti proliferative activity against human colon cancer cell HT-29, HCT116 and HCT-15, cervix HeLa cell, breast cancer cells MDA-MB 468 and MCF-7. From the results, it can be concluded that the pulp part has potential to be used in the treatment of cancer [51].

Apoptosis is a physiological procedure to maintain homeostasis in healthy tissue and suppression of damaged cells. There are many chemopreventive agents, which result in cancer cell death by induction of apoptosis [52].

In this section flavonoids induced cellular cytotoxicity through apoptotic signalling, cell cycle regulation mechanisms by one of the major classes of flavonoids, i.e., quercetin.

FITC Annexin V staining follows the loss of membrane integrity which attends the latest stages of cell death resulting of the apoptotic processes. Thus, staining with FITC Annexin V is normally used in combination with a vital dye such as propidium iodide (PI) or 7-amino-actinomycin (7-AAD) to allow the investigator to detect early apoptotic cells. In the present study, the effect of A. altilis fruit extract on apoptosis, was distinguished from living cells with early apoptotic cells by Annexin V-FITC+/7-AAD- and late apoptotic cells by Annexin V-FITC+/7-AAD+ on selected cancer cell line in a time dependent manner. Acquired data results were displayed on the computer screen in a dot-plot format with a user-controlled quadrant marker (Figure 3a). This was further confirmed by a flow cytometric analysis using annexin V and 7-amino-actin (7-AAD) double staining. Recent report by Sun et al. mentioned that quercetin induced apoptosis in both the human breast cancer (SK-BR-3) and the human epidermoid carcinoma (A-431) cell lines with concentrations starting with 50 and 75 μg/mL [53]. While in gastric cancer cell lines (BGC-832) the concentration was 90 or 120 μg/mL.

Quercetin led to increase in CASPASE-3 and BAX and reduction in BCL-2 expression [54]. Numerous mechanisms can explain the ability of quercetin to induce apoptosis. It has been reported that apoptotic effect of quercetin in colon and breast cancer cells was allied to the ability to generate reactive oxygen species which in turn could lead to DNA damage resulting in cell death Zhang et al. [55]. As stated that quercetin induced G0/G1, G2/M and S phases arrest followed by apoptosis in in three leukemic cell lines (CEM, K562 and Nalm6), two breast cancer cell lines (T47D and EAC) and colorectal carcinoma Yuan et al. Yoshida et al. Richter et al. [56-58]. Previously reported in this study that the contain of quercetin in pulp 78%.

Hartwell and Kastan reported that the induction of apoptosis might be due by the inhibition of DNA replication caused by the inability of the cells to replicate damaged DNA caused by the plant extracts [59]. Jangi et al. Poon et al. reported that when cells arrested at G2/M checkpoint and prevent entry into mitosis due to incomplete replication of DNA [60,61]; it has been shown that quercetin treatment caused cell cycle arrests such as G2/M arrest or G0/G1 arrest in different cell types. Khan et al. demonstrated that A549 cells were arrested at G2/M phases by pulp, which was associated with induction of p21 and controlled by decrease in the expression of downstream cell cycle regulatory proteins [62]. Singh et al. [63], Janicke et al., Choi and Kim mentioned that type of phenolic acids causes cells arrest at G2/M such as quercetin, gallic and ferulic acids. As chemopreventive agents, flavonoids are active at different stages of cancer development, interfering with the overall process through various mechanisms such as up/ down regulation of apoptotic and cell cycle genes.

Flavonoids act to inhibit the activity of DNA topoisomerase I/II (Snyde and Gillies) [64], release of CYTOCHROME C from mitochondria and following activation of CASPASES-3, 8 and 9 (Michels et al.; Wang et al.) downregulate BCL-2 expression and upregulate BAX expression leading to apoptosis Wang et al. [65,66]. Quercetin is known as one of the major classes of flavonoids and in this study it was found that pulp contained high quantity of quercetin which resulted in pulp mediating downstream intrinsic pathway but with high fold of gene expression change. Fadeel and Orrenius mentioned that once BAX was activated, it promotes CYTOCHROME C release and mitochondrial breakdown [67]. ATP in turn release cytokines which suppress the inflammatory response, which triggers the activation of APAF1 into an apoptosome activating the CASPASE-9 molecules (upon cleavage of the bound zymogen procaspases-9). This in turn activated CASPASE-3 and CASPASE-9 Vodovotz et al. and Wei et al. demonstrated that tBID activates BAX molecules which leads to the release of CYTOCHROME C from the mitochondria. BAX has been shown to be unnecessary in tBID induced CYTOCHROME C release [68,69]. A heme-like structure containing protein CYTOCHROME C located on the outside of the inner mitochondrial membrane (proteins control mitochondrial membrane permeability) binds to Apaf-1 which is released into the cytosol. Together with ATP it binds to apoptotic protease activating factor (Apaf1) and forms the apoptosome complex Elmore Los et al. [70,71]. Makiuchi and Nozaki stated that mitosome is a highly-reduced form of mitochondria that do not contain any genome and have lost the capacity to generate energy [72]. CASPASE-9 becomes activated in the apoptosome and then activates CASPASE-3. Wei et al. and Emily et al. reported that the intrinsic pathway which is also called the mitochondrial pathway, is regulated by Bcl-2 family of proteins (BH, BCL2-homology). Induction or post-translational activation resulted in the significant inhibition of the expression of BCL-2 in all treated cell lines used in this study [69,73].

It also resulted in changes in cell cycle phase’s inactivation of some BCL-2 family members. Up and down regulation of cell cycle gene expressions will lead to clear figures of the downstream arrest of treated cancer cell lines induced by the fruit extracts stimuli. A number of studies reported that involvement of cyclins and CDKs are important for regulation of cell cycle checkpoints in eukaryotes [74]. Marinelli et al. mentioned that a large number of studies have shown that p21 plays a role in tumour suppression in cancer [75]. P21 can promote apoptosis depending on particular cellular stresses when it results in upregulation and is a common CYCLIN-CDK complex inhibitor that is activated in response to different stress stimuli and could act as cell cycle suppressor [76].

This study demonstrated that the molecular mechanisms for A. atilis fruit extract induced cell growth inhibition and the occurrence of apoptosis in selected human cancer cell lines. Furthermore, it shows that the mode of action of the cell death signalling can be separated into two pathways. Volate et al. reported that quercetin can induce apoptosis via the mitochondrial pathway as against the harmful effects of carcinogenic chemicals by offering a defence system [77], by damaging DNA and initiating pro-oxidant property in cells as in A549 and HT- 29 cells [78]. Lee et al. reported that CDK1/CYCLIN B1 complexes promote the transition in mitosis through the phosphorylation of some substrates as APC (Anaphase Promoting Complex) [79], responsible for the transition from metaphase to anaphase. Finally, p21 can act on G2/M checkpoint through the downregulation of Emi1 (Early mitotic inhibitor).

Emi1 regulates mitosis by inhibiting APC/C ubiquitin ligase complex (Anaphase Promoting Complex/Cyclosome) during S and G2 phases allowing a correct mitotic entry and preventing re-replication. It has been shown that Emi1 repression results in APC/C-dependent degradation of CYCLIN A1 and B1 and G2 phase arrest activated for the mitosis transition. Charrier-Savournin et al. suggested that p21 can block this progression by binding the complex and interfering with CDK1 dephosphorylation on Thr14 and Tyr15; and with CYCLIN B1 phosphorylation on Thr161 [80]. Therefore, pulp arrested the treated cells at G2/M resulting in disruption of normal chromatid disjunction during mitosis in mammalian cells and disrupting the breaking of the chromosome by the mitotic spindle during mitosis. Tumour cell lines increase sensitivity to anticancer agents. For example, several flavonoids, such as quercetin which had been shown to inhibit tumour growth in animal models were able to interrupt cell cycle progression in either G1 or G2 phase. The current studies indicate that polyphenols induction of cellular cytotoxicity may be occurring through a nonclassical apoptotic mechanism. It also showed that flavonoids are cytotoxic to cells, which lack CASPASE-3 and CASPASE-8. However, more extensive research into the role of flavonoids in each signalling pathway is still required. These findings provide potential effectiveness and a theoretical basis for the therapeutic use of A. altilis fruit extracts in the treatment of malignancies. A. altilis fruit extracts induced cellular apoptosis, apparently by acting on the functions of many intracellular genes important for these apoptotic process. In addition, comparing the apoptotic mechanisms of the three parts of the fruit on four cancer cell lines, was found to be a potent inhibitor of apoptosis in response to a variety of cytotoxic stimuli.

Conclusion

One of the remarkable results of the present work was to identify and quantify some phenolic compounds present in the methanol extracts of A. altilis, by using the UHPLC-MS/MS based approach. Nine phenolic compounds were present, broadly identified as flavonoids. Phenolic acids were detected and characterized on the basis of their chromatographic retention time, UV-vis spectra and mass spectra in the negative-ion mode and data from the literature. Overall, pulp presented the highest concentrations of total flavonoids and phenolic acids, with rutin and protocatechuic acid being the major compounds.

Quercetin and protocatechuic acid were the most abundant flavonoid and phenolic acid quantified in the pulp part. Indeed, the results indicate qualitatively and quantitatively that A. altilis fruit is an abundant source of phenolic compounds for use in the food and pharmaceutical industries. The fruit extracts inhibited the growth of the cells through induced apoptosis and cell cycle arrest at G0/G1, S and G2/M. Quercetin significantly inhibited the expression of particular tumour growth and angiogenesis by inducing apoptosis and regulating at G(1), S, G(2), and M phases of the cell cycle. Additionally, it has been shown to upregulate the expression of several tumour suppressor genes, via initiation of tumour suppressor genes. Inhibition of expression of tumour cell activity inhibited the cell cycle and reducing the risk of cancer occurrence. The methanol extract of pulp of A. altilis fruit inhibited the proliferation of selected cancer cells in vitro by causing cell cycle arrest at G2/M inducing apoptosis mediated, at least in part, via CASPASE-3 and mitochondrial apoptosis pathways in both caspasedependent and independent manner. The molecular mechanisms involved are the up-regulation of the BAX/BCL-2 ratio, the activation of CASPASE-8 and CASPASE-3. The apoptosis were mediated by pulp via mitochondria pathway on A549 cells. The variation in the mechanism of the three parts of the fruit-induced cell cycle arrest and apoptosis in selected cancer cell lines may be dependent on the genetic profile of the cells.

Conflict of Interest

None.

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