Evaluation of Anti-inflammatory and Anti-proliferative Activity of Abutilon indicum L. Plant Ethanolic Leaf Extract on Lung Cancer Cell Line A549 for System Network Studies

Pharmaceutically important components in medicinal plant kingdom are useful to control and cure many diseases like lung cancer. In the present study, an evaluation was performed to know the anti-inflammatory and anti-proliferative activity of ethanolic leaf extract of Abutilon indicum for potential chemopreventive agent against lung cancer. Experimentation is also conducted on lung cancer cell line A549 along with interaction studies of Apaf1 gene. The ethanolic leaf extract of A. indicum is showing good anti-inflammatory activity (IC50: 8.89 μg/mL) based on 5-Lipoxygenase (5-LOX) inhibition assay. The standard compound, Curcumin has shown IC50 as 8.14 μg/mL based on study. The ethanolic leaf extract of A.indicum has also shown good response on human Caucasian lung carcinoma of A549 cell line (IC50: 85.2 μg/mL) shows anti-proliferative activity. Further analysis has also shown the interaction of proteins that are involved in inflammatory and cancer response. Apoptosis-activating factor, Apaf1 gene increases the sensitivity of A549 cell line through interaction with proteins like CASP9, CASP3, CYCS, BCL2L1, TP53, BCL2, CASP8, HSPA4, DIABLO and CASP7. The experimental work concludes that bioactive components present in the ethanolic leaf extract of A.indicum shows good anti-inflammatory and anti-proliferative activity by inducing Apaf-1 through CASP9, CASP3, CYCS, BCL2L1, TP53, BCL2, CASP8, HSPA4, DIABLO and CASP7 network. Citation: Kaladhar DSVGK, Saranya KS, Vadlapudi V, Yarla NS (2014) Evaluation of Anti-inflammatory and Anti-proliferative Activity of Abutilon indicum L. Plant Ethanolic Leaf Extract on Lung Cancer Cell Line A549 for System Network Studies. J Cancer Sci Ther 6: 195-201. doi:10.4172/1948-5956.1000271 Volume 6(6) 188-194 (2014) 196 J Cancer Sci Ther ISSN:1948-5956 JCST, an open access journal the cellular system in lungs will be damaged and mutations may occur leading cancer [11,12]. Most of the cases for the cancer deaths worldwide are due to lung cancers that are associated with cigarette smoking. Human lung cancer cell line A549 may provide a molecular understanding of ethanolic extract of Abutilon indicum that blocks cell cycle progression in the G1 phase. The association and networks may be due to activating partner cdk2, 4 and 6 that induce p21/WAF1/CIP1, p300/CBP and Apaf1. The effect is also due to up-regulation of Bax, Fas ligand and Fas/APO-1 and in down-regulation of NF-κB, Bcl-2, and Bcl-X (Figure 1). MAPK pathway Controlling apoptosis in cancer development can be a major barrier to provide effective treatment using phytocompounds. Figure 2 shows Mitogen-Activated Protein Kinase (MAPK) cascade is involved in various cellular functions like cell proliferation, differentiation, apoptosis, migration and cancer [13]. This is a highly conserved module that expresses extracellular signal-related kinases like (ERK)-1/2, Jun amino-terminal kinases (JNK1/2/3), p38 proteins and ERK5 that are activated by specific MAPKKs. The MEK1/2, ERK1/2, MKK3/6, p38, MKK4/7 (JNKK1/2), JNKs, MEK5, ERK5 forms networks in mammals are linked to inflammation and cancer. Each MAPKK in turn be activated by more than one MAPKKK showing increase in complexity and diversity of MAPK signalling. MAPKKKs may activate ERK1/2 in response to pro-inflammatory stimuli (Figure 2). The present investigation attempted to  reveal the antiinflammatory and anti-cancer activity of A.indicum which was used as an ethnomedicine in India and several other parts of the world. Understanding of the Apaf1 protein network can be helpful in cracking the role of Apaf1 in cancer development and conventional therapy. Protein interaction networks can be performed to know interaction between cancer and inflammation causing proteins to establish the relationship within and outside the cell. Materials and Methods Preparation of extracts The A. indicum leaves were collected from Visakhapatnam regions during rainy season. The plant is authenticated by Dr. P.V. Arjun Rao, Ethanobotanist, Dept. of Botany, Phytopharma Technology Laboratory, GITAM University, Visakhapatnam (No. Res/3). The leaves were dried and the powder was prepared. Nearly 20 grams of the powder was soaked in 200 ml of ethanol for 24 hours in rotary shaker, filtered and centrifuged for 30 min at 4000 rpm. The supernatant solution was collected after centrifugation and was dried in the sunlight for getting powder. After drying 2 grams (equal to 10% yield) has been obtained. The powder was used for the present experimentation. Anti-inflammatory activity 5-Lipoxygenase (5-LOX) is an important enzyme involved in inflammation. The anti-inflammatory effect refers to the property of a treatment or a substance to reduce inflammation that inhibits 5-LOX. The standard used in the present experiment is Curcumin. The test sample used in the experiment is ethanol leaf extract of A. indicum. Appropriate dilutions (70 μg/mL-1 μg/mL) of test items were prepared to assess the 5-LOX activity. Chemicals and reagents All the chemicals acquired from Sigma chemicals and the reagents are prepared as per the below given protocol. SMALL CELL LUNG CANCER Pulmonary neuroendocrine epithelial cell Neuroendocrine epithelial cells Reduced apoptosis Cell-cycle progression Retinoic acid p53 signaling pathway Overexpression


Introduction
Plants have invariably had typical constituents of medicaments used from ancient preparations or as pure active principles. Plant extracts contains important sources of drugs used in control of aging and diseases. There are specific plant structures used as crude drug substances as active or major chemical constituents against diseased proteins [1]. These chemical components show characteristic profile that may be used to control networks in a system. The drug constituents with scientific data measure delineate within the major chemical constituents on the efficacy, security, beneficiary and internal control is used from plants which are associated with protein targets within a system.
Ayurveda is an ancient and applied research area used for the utilization of plants in treatment of varied human ailments. India has regarding 45,000 plant species and among them many are claimed to possess healthful properties [2]. Medicinal plants of the world contain valuable species that have indigenous, naturalized or cultivated plants [3]. Many Indian and Chinese healthcare plants historically employed in medicines were subjected to preliminary medicinal drug screening against morbific and opportunist microorganisms [4,5].

Abutilon indicum
Abutilon indicum (Linn.) Sweet (Malvaceae) is a shrub distributed throughout India, Sri Lanka, topical regions of America and Malaysia [6]. It is known as Atibala in Sanskrit. It grows as weeds that are found abundant in wastelands and seashores.
The plant has a large number of pharmacological activities and there are no reports on the exact mechanism of plant ethanolic leaf extract of A.indicum for activity against lung cancer A549 cell line. From the ancient times, leaves of A.indicum are used for lumbago, piles, toothache, and all kinds of inflammation. Decoction of A.indicum leaves is used in catarrhal bilious diarrhoea, bronchitis, gonorrhoea, fevers and inflammation of bladder [6]. The decoction is prescribed as a mouthwash in various cases of tender gums and toothache. Bark of A.indicum is used in strangury and urinary complaints and is valued as a diuretic. Seeds are used as tonic. Unani systems of medicine suggest use of seeds in piles, chest troubles, bronchitis, and gonorrhea. Rectum of children affected with thread worms can be cured by exposure to the smoke of seeds burned on charcoal. According to the Chinese from Hong Kong, the seeds are employed as an emollient and demulcent [5].
the cellular system in lungs will be damaged and mutations may occur leading cancer [11,12]. Most of the cases for the cancer deaths worldwide are due to lung cancers that are associated with cigarette smoking. Human lung cancer cell line A549 may provide a molecular understanding of ethanolic extract of Abutilon indicum that blocks cell cycle progression in the G1 phase. The association and networks may be due to activating partner cdk2, 4 and 6 that induce p21/WAF1/CIP1, p300/CBP and Apaf1. The effect is also due to up-regulation of Bax, Fas ligand and Fas/APO-1 and in down-regulation of NF-κB, Bcl-2, and Bcl-X ( Figure 1).

MAPK pathway
Controlling apoptosis in cancer development can be a major barrier to provide effective treatment using phytocompounds. Figure 2 shows Mitogen-Activated Protein Kinase (MAPK) cascade is involved in various cellular functions like cell proliferation, differentiation, apoptosis, migration and cancer [13]. This is a highly conserved module that expresses extracellular signal-related kinases like (ERK)-1/2, Jun amino-terminal kinases (JNK1/2/3), p38 proteins and ERK5 that are activated by specific MAPKKs. The MEK1/2, ERK1/2, MKK3/6, p38, MKK4/7 (JNKK1/2), JNKs, MEK5, ERK5 forms networks in mammals are linked to inflammation and cancer. Each MAPKK in turn be activated by more than one MAPKKK showing increase in complexity and diversity of MAPK signalling. MAPKKKs may activate ERK1/2 in response to pro-inflammatory stimuli ( Figure 2).
The present investigation attempted to reveal the antiinflammatory and anti-cancer activity of A.indicum which was used as an ethnomedicine in India and several other parts of the world. Understanding of the Apaf1 protein network can be helpful in cracking the role of Apaf1 in cancer development and conventional therapy.
Protein interaction networks can be performed to know interaction between cancer and inflammation causing proteins to establish the relationship within and outside the cell.

Preparation of extracts
The A. indicum leaves were collected from Visakhapatnam regions during rainy season. The plant is authenticated by Dr. P.V. Arjun Rao, Ethanobotanist, Dept. of Botany, Phytopharma Technology Laboratory, GITAM University, Visakhapatnam (No. Res/3). The leaves were dried and the powder was prepared. Nearly 20 grams of the powder was soaked in 200 ml of ethanol for 24 hours in rotary shaker, filtered and centrifuged for 30 min at 4000 rpm. The supernatant solution was collected after centrifugation and was dried in the sunlight for getting powder. After drying 2 grams (equal to 10% yield) has been obtained. The powder was used for the present experimentation.

5-Lipoxygenase (5-LOX) is an important enzyme involved in
inflammation. The anti-inflammatory effect refers to the property of a treatment or a substance to reduce inflammation that inhibits 5-LOX. The standard used in the present experiment is Curcumin. The test sample used in the experiment is ethanol leaf extract of A. indicum. Appropriate dilutions (70 µg/mL-1 µg/mL) of test items were prepared to assess the 5-LOX activity.

Chemicals and reagents
All the chemicals acquired from Sigma chemicals and the reagents are prepared as per the below given protocol.

Assay mixture
About 250 µL of assay mixture prepared that contains 20 µL test sample of different concentrations dissolved in 175 µL of 50 mM Tris-HCL buffer, 5 µL enzyme, 5 µL Lenoleic acid and 65 µL FOX reagent was prepared.

Experimental method
Approximately 5 µL of 5-LOX enzyme was added with 175 µL of 50 mM Tris HCL buffer (Pre-incubated with 20 µL test sample for 5 min at 25ºC) is to be prepared. The reaction is then initiated by addition of 5 µL linoleic acid (final concentration 140 µM) in 50 mM Tris HCL buffer followed by incubation at 25ºC in dark for 20 min. The above total volume contained 185 µL of reaction mixture is terminated by the addition of 65 µL freshly prepared fox reagent. After the termination the absorbance was taken at 595 nm upto 30 minutes at 25ºC with Xmark Micro plate spectrophotometer (BIO-RAD).

In vitro anti-proliferative activity
Medicinal plants acts as useful source in research for finding new biologically active compounds, especially for ethno-medical data approach. The ethno-medical data is not always completely reliable as it is difficult to diagnosed and treat cancer.
Caucasian lung carcinoma of A549 cell line used in this study is procured from National Centre for Cell Science (NCCS), Pune. The cells were grown in Minimal essential medium (MEM, GIBCO) that is supplemented with 4.5 g/L glucose, 2 mM L-glutamine and 5% fetal

XTT assay
The biochemical procedure is based on the activity of mitochondrial enzymes which are inactivated shortly after the cell death. The method is found to be very efficient in considering viability of the cells. Colorimetric based method based on the tetrazolium salt, XTT. The trypsinized cells from T-25 flask were seeded in each well of 96well flat-bottomed tissue culture plate at a density of 5x10 3 cells/well in growth medium and cultured at 37°C in 5% CO 2 to adhere. After 24 hrs incubation, the supernatant was discarded and the cells were pretreated with growth medium. The media were subsequently mixed with different concentrations of test compounds (12.5,25,50,100 and 200 µg/ml) to achieve a final volume of 100 µl were cultured for 48 hrs. The compound is prepared as 1.0 mg/ml concentration stock solution prepared with in DMSO solution. Culture medium and solvent are used as controls. Each well then received 50 µl of fresh XTT (0.9 mg/ ml in RPMI along with XTT activator reagent) followed by incubation for 2 hrs at 37°C. At the end of the incubation, shake the 96 micro well plates for 15 seconds on a shaker. The Optical Density (OD) of the culture plate was read at a wavelength of 490 nm (reference absorbance at a wavelength of 630 nm) on Anthos 2020 spectrophotometer.

Protein interaction network mapping
Apaf-1 is a vital factor responsible for activation of cytochrome c-driven caspase during the mitochondrial apoptosis. It is an important tumor suppressor like lung carcinoma of A549 cell line. Protein interaction network mapping was constructed and analysed for mechanism using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) 9.05 server used to know and predict protein interactions. In the present work, "Apaf-1" was selected as query protein to understand the interaction network between cancer and inflammatory causing proteins.

Results and Discussion
The plant products like glycosides in A. indicum may play an important role in the decreased risk of chronic diseases associated with a diet and a wide range of disease [14]. The studies on cytotoxic effects of some anti-cancer drugs suggest that phytocompounds has cancer chemo-therapeutic and chemo-preventive potential without side effects [15,16]. 5-LOX and cyclooxygenase (COX) inhibition may have anticarcinogenic effects. COX and LOX inhibitors that are used for the treatment of inflammatory diseases act as potential anticancer drugs [17][18][19]. The ethanolic leaf extract of A. indicum has shown good anti-inflammatory Activity (IC 50 : 8.89 µg/mL) based on 5-LOX assay. The IC 50 values of test sample and the standard are 8.89 µg/mL and 8.14 µg/mL respectively ( Figure 3 and Table 1). A. indicum extract has not shown much variation with standard Curcumin. The isolated samples may further show good phytochemical components for antiinflammation and anti-cancer activity. These results have provided that the plant extract has shown good anti-inflammatory activity and may provide the involvement in control of disease like cancer.       of A.indicum showed good anti-proliferative activity, less compared to standard at IC 50 . Figure 5 shows antiproliferative response of ethanol leaf extract of A.indicum against Lung cancer cells (A549) demonstrated in before and after treatment of extract. There is more number of lung cancer cells before the treatment with plant compound. After adding 200 µg/ mL of ethanolic leaf extract of A.indicum has shown shrinkage/lysis in lung cancer cells.
Cell growth inhibition (LD 50 ) on HeLa cells for an ethanolic extract of saffron (Crocus sativus L.) dry stigmas was shown as 2.3 mg/ml [20]. Ethanol extracts of S. barbata was inhibited A549 cell growth (IC 50 ) of 0.21 mg/ml [21]. An ethanol intake was not been linked with lung cancer [22] and was not having effect on NNK metabolism [23] in mouse lung microsomes. Hence the intake of ethanol may cause lung cancer but the ethanolic extracts contains phytocompounds that are non-toxic and shown antiproliferative activity on A549 (Lung cancer cell line).
Molecular interaction studies provide better understanding component stacks in lung cancer towards drug discovery and designing. Silencing of miR-155 and the over expression Apaf-1 greatly increased the sensitivity of A549 cells involved in lung cancer to cisplatin [24]. Figure 6 was provided the mechanism of Apaf1 interaction with other proteins in link to lung cancer using string database. Apaf-1 has shown interactions with the proteins like CASP9, CASP3, CYCS, BCL2L1, TP53, BCL2, CASP8, HSPA4, DIABLO and CASP7. These components has shown network with components relevant to MAPK signaling pathway that links to inflammation and lung cancer.
In the present decades, plant kingdom is considered as gold mines due to existence of many biologically active principles that show therapeutic value [25]. Cancer is a major public health problem in all around the world that does not contain complete cure [26]. The interfering lung epithelial cell death using phytocompounds is an important protective and therapeutic strategy [27]. The ethanolic leaf extract of A.indicum contains components that show antiproliferative response for lung cancer cell line (A549) through Apaf-1 gene by activation of cytochrome c-driven caspase like CYCS etc.
A. indicum leaves extract enhances the antioxidant potential due to free radical scavenging activity [28,29]. A. indica leaf extract was found to possess better antioxidant activity may be due to the phenolics and flavonoids present in the extract. The F. hispida and A Scholaris leaf extracts in ethanol exhibited more cytotoxicity when compared to other extracts in HeLa and MCF-7 cell lines [30]. The present experimentation has shown good anti-oxidant and antiproliferative activity of ethanol extract of A.indicum and shown interactive mechanism with MAP Kinase pathway. Protein network analysis provided molecular understanding showing network topologies in the biological systems that regulates anti-apoptotic molecules and cell cycle progression [31].

Conclusion
The ethanolic leaf extract of A.indicum has shown medicinal importance with anti-inflammatory and anti-proliferative properties. The study has shown interactive mechanism with MAP Kinase pathway. There may be future scope to discover the novel anti-inflammatory and anti-cancer drugs from ethanolic leaf extract of A. indicum.