INTRODUCTION:

Cancer is the second most widespread cause of death, surpassed only by coronary heart disease. Additionally, in India the overall cancer cases are expected to go up from 979,786 cases in the year 2010 to 1,148,757 cases in the year 2020 (Takiar 2010). Lung cancer is the first in rank cancer killer in the United States of men and women of all ethnicities. Lung cancer kills more people in the United States every year than breast, colon, and prostate cancer combined. Lung cancer is mostly a disease of the elderly. Lung cancer prospers when the cells that line the lungs preserve genetic damage.

More than half of lung cancers happen in people who are as choice current or former smokers. Epidemiological and experimental studies provide confirmation that some naturally occurring chemical agents in the human diet can reduce cancer risk. Plants produce diverse arrays of phytochemicals which are useful in the development of new drugs. Phytochemicals are natural and non-nutritive bioactive compounds produced by plants that act as protective agents against external stress and pathogenic attack. In-vivo and in vitro studies also documented the role of several phyochemicals in cancer prevention. Some naturally occurring chemical agents in the human diet can reduce cancer risk such as, Lupeol also known as fagarsterol [Lup-20(29)-en-3β-ol], a diet-based triterpene,is the principal constituent of mango fruit (Mangifera indica; Family- Anacardiaceae) and others e.g. olive, strawberry, grapes, figsetc. has been reported to possess a wide range of medicinal properties that include strong antioxidant, antimutagenic and anti-inflammatory effects.Several in-vitro studies provide insight into the mechanism of action of lupeol and suggest that it is a multi-target agent with immense anti-inflammatory potential targeting several key molecular pathways which involve COX-2, NF-κB, survivin/cFLIP, K-ras, PI3K/Akt and Wnt/β-catenin in cancer cells. Lupeol is reported to activate apoptotic machineryby means of both Fas signaling (Saleem et al .,2004) and mitochondrial mode (Prasad et al 2008). Its treatment in 451Lu melanoma cells caused G(1)-S phase cell cycle arrest and decreased the expression of protein cyclin D1, D2, and Cdk2; and increased the expression of p21 . In-vitro lupeol inhibits the tumorigenicity of androgen-sensitive prostate cancer cells with a concomitant decrease in serum PSA levels under in vivo conditions (Saleem et al .,2004). It reduces the proliferative and clonogenicpotential of androgen-sensitive as well as androgen-insensitiveprostate cancer cells by modulating β-catenin-signaling pathway. The treatment of cells with a combination of anti-Fas monoclonal antibody and lupeol resulted in higher cell death when compared with alone (Saleem et al .,2005). Employing a focused microarray of human prostate cancer associated genes, found that lupeol significantly modulates the expression level of proliferation and survival associated genes such as ErbB2, TIMP-3, cyclin D1 and MMP-2, which are known to either regulate or act as downstream target of β-catenin signaling. In addition lupeol induced growth inhibition of prosate cancer cells is an outcome of disruption of microtubule assembly through simultaneous effect on stathmin, cFLIP, and survivin molecules .Lupeol potential in prevention of cancer are proved by in vivo studies. Lupeol treatment inhibits head and neck cancer in a mouse tumor xenograftmodel and exerted a synergistic effect with cisplatin . Lupeol-induced G2/M-phase arrest was mediated through inhibition of the cyclin-B-regulated signaling pathway involving p53, p21/WAF1, Cdc25C, Cdc2, cyclin-B and Plk-1 in skin cancer model (Nigam et al., 2007). Lupeol/mango pulp extract supplementation resulted in inhibition of prostate enlargement in testosterone-treated animals (Prasad et al 2008). Thus, lupeol could be a potential agent against cancer; further in-depth studies are warranted to allow the therapeutic application of this phytochemical. It is noteworthy that Lupeol at its effective therapeutic doses exhibit no toxicity to normal cells and tissues. Our study focuses to determine the cancer chemotherapeutics potential of Lupeol for the treatment of lung cancer.

MATERIALS AND METHOD:

Human epithelial carcinoma A549 cells were obtained from National Centre for Cell Science (Pune, India) and cultured in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% heat inactivated fetal bovine serum, penicillin streptomycin (Gibco Lifetech, Karlsruche, Germany).The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 inside a CO2 incubator.

Cell Culture and Treatment:

Cancer chemotherapeutics aspects of lupeol were evaluated in in vitro test system by using lung cancer cell line A549.Switch on the UV lamp 40min prior to media preparation.

Treatment protocol

Lupeol stock solution was prepared at 1mM concentration in minimal amount of DMSO and diluted in fresh medium to achieve desired final concentration for treatment of cells. For dose dependent studies, the cells (80% confluent) were treated with lupeol (50, 75 and 100 μM) for 24 h, 48 h, 72 h and 96 h, respectively, in complete cell culture medium. Cells that served as controls were incubated with the vehicle (DMSO) only. The final concentration of DMSO was 0.2% in all treatment protocols. The cells were harvested by trypsinization, washed twice with cold phosphate-buffered saline (PBS) and stored at -80°C (New Brunswick Scientific, Germany) till further analysis.

Concentration (µg/ml)	suspension (µl) (From the stock)	Media (CDMEM F-12) (µl)
50	120	2280
75	180	2220
100	240	2160

Cell Viability Assay

MTT assay:

The effects of treatments on the viability of lung cancercells was determined by MTT assay (Mosmann, 1983). The IC₅₀ value was determined by plotting a graph of percentage considered of survival versus lupeol concentrations where cells with no treatment were considered to be 100% viable. MTT assay is an important colorimetric assay to assess cytotoxicity based on the metabolic activity of the viable cells. Yellow MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] is reduced to a purple colored formazan product in living cells by the “succinate-tetrazolium reductase” system (EC 1.3.99.1) that belongs to the mitochondrial respiratory chain (Fig. ) and is active only in viable cells (Mosmann,T, 1983). Interestingly however, recent evidence suggests that mitochondrial electron transport may play a minor role in the cellular reduction of MTT. Since most cellular reduction occurs in the cytoplasm and probably involves the pyridine nucleotide cofactors NADH and NADPH, the MTT assay can no longer be considered strictly a mitochondrial assay. For example, a micro titer plate assay, which uses the tetrazolium salt MTT is now widely use to quantitate cell proliferation and cytotoxicity (Morgan, D.O., 1997). Because tetrazolium salts are reduced to a colored formazan only by metabolically active cells containing active reductase system, these assays detect viable cells exclusively. This water insoluble formazan salt can be solubilized in usually either dimethyl sulfoxide (DMSO), an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid and absorbance of the colored solution can easily and rapidly be quantitated in a conventional ELISA plate reader at wavelength between 500 to 600nm (absorbance maximum depending upon the solvent used).

More recently, modified tetrazolium salts like XTT, MTS and WST-1 have become available. The major advantage of these compounds is that viable cells convert them to a water soluble formazan. Thus, a metabolic assay with any of these compounds requires one less step ( solubilization of product than an assay with MTT. Freshly isolated A549 (1 x 10⁴/100 ml per well) were plated in 24 well plate.Cells were incubated for 24, 48, 72 and 96 hrs in a humified CO₂incubator at 37°C.10 ml of MTT was added to each well 4 hours before the completion of the time period.The medium was removed and 100 ul DMSO was added to solubilize the fomazone crystals.The absorbance was read in an ELISA plate reader at 530 nm.The results are expressed as percentage of control.

Flow-cytometry analysis of apoptosis

Cells were fixed in chilled ethanol (70%) and incubated with PI (50 µg/ml) for 30 min at 4⁰C. The cells were acquired and analyzed for studying cell cycle parameter using a flow-cell cytometer (Becton–Dickinson LSR II, San Jose, CA, USA) and ‘Cell Quest’ software.

Flow cytometric analysis of ROS level

ROS production was monitored in A549cells by flow cytometry (BD-LSR II, San Jose, CA, USA) using dichlorodihydrofluroscein diacetate (DCFH-DA) dye as described by Degli Esposti and McLennan (1998). The fluorescence, increased due to the hydrolysis of DCFH-DA to dichlorodihydrofluroscein (DCFH) by some nonspecific cellular esterases and its subsequent oxidation by peroxides, will be measured. Values will be given in terms of mean fluorescence intensity (MFI) using software ‘cell quest’.1.5x10⁶/ml freshly isolated A549 were plated in DMEM medium. The cells were treated with various concentrations. of Lupeol (50µM, 75µM, 100µM) and incubated for 24,48,72 and 96 hrs in CO₂incubator at 37^oC .The cells were collected, resuspended in 0.25 ml PBS and fixed with 0.5 ml chilled absolute alcohol (final conc. 70%) and kept on ice for approximately one hour.Fixed cells were centrifuged at 2000 rpm for 5 minutes at 4°C and washed with 0.5 ml PBS. The cells were resuspended in 0.5 ml PBS and 0.1ml phosphate citrate buffer and incubated for one hour at room temperature. Cells were centrifuged again and 0.25 ml PI solution (10 mg PI, 0.1 ml Triton – X 100 and 3.7 mg EDTA in 100 ml PBS) and 0.25 ml RNase solution (stock 50 mg / ml) were added. Samples were incubated again for 30 minutes in dark. The PI fluorescence was measured through a FL-2 filter ( 585nm) and 10,000 events were acquired .The cell population coming before G_o /G₁ phase i.e. sub G₁ represents apoptotic DNA

Mitochondrial membrane potential

Mitochondrial membrane potential was determined by using Rhodamine 123 dye. Rh 123, a lipophilic cationic fluorescent dye, is selectively taken up by mitochondria and its uptake is directly proportional to mitochondrial Dy of cells .1.5 x 10⁶ /ml A549 cells were plated, treated with various conc. of lupeol (50µM, 75µM, 100µM) and incubated for 24,48,72 and 96 hrs in CO₂incubator at 37^oC .The cells were collected and centrifuged at 2000 rpm for 5 minutes.The supernatant was discarded and cells were resuspended in 0.5 ml of PBS. Rh 123 (10μl) was added and further incubated for 30 min at 37°C.The fluorescence was acquired at FL-1 filter on BD-LSR Flow cytometer. Cell debris, characterized by a low FSC / SSC was excluded from analysis. The data analysed by Cell Quest software and mean fluorescence intensity was obtained by histogram statistics.

Oxidative stress markers

Assessment of ROS generation:

The generation of ROS was detected by using DCFH-DA dye. DCFH-DA, a permeable dye, is cleaved to form non fluorescent dichlorofluorescein (DCFH) in the cells. It gets oxidised to fluorescent dichlorofluorescein (DCF) by ROS. The DCF fluorescence is proportional to the ROS levels in the cells which was monitored on flow cytometer.

Protocol: A549 cells were plated, treated with various conc. of lupeol (50µM, 75µM, 100µM) and incubated for 24, 48, 72 and 96 hrs in CO₂incubator at 37^oC.DCFH-DA (100μM) was added and further incubated for 30 min at 37°C. cells were collected and centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded and cells were resuspended in 0.5 ml of PBS. Fluorescence was acquired at FL-1 filter on BD-LSR Flow cytometer. Cell debris, characterized by a low FSC / SSC was excluded from analysis. The data analysed by Cell Quest software and mean fluorescence intensity was obtained by histogram statistics.

Protein estimation prior to electrophoresis

Prior to Western blotting the protein content in the sample was estimated by Lowry’s method.

First standard curve of bovine serum albumin (BSA) was made, for this 10mg/ml stock solution of the BSA was prepared. 10,20,30,40,50μg of BSA stock solution was used, which was then made-up to 100µl with double distilled water 5ml Alkaline Reagent (2%Na₂CO_3,0.4%NaOH, 1%CuSO_4,2% Sodium potassium tartarate) was added in each tube and vortexed. 500 µl of Folin’s reagent was added to each tube and incubated at room temperature for 35 min in dark. Absorbance was measured at 660nm.The concentration of the protein in the sample was calculated from the standard curve of BSA.

SDS-PAGE

10μg, 20μg, 40μg, 80μg were mixed with gel loading dye (50mM Tris.Cl (pH-6.8),0.1% BPB 10% Glycerol ) and 1M DTT and heated at 100ºC on dry bath for 2min each followed by centrifugation at 10,000 rpm for 5min. The samples were loaded on 5% stacking gel (pH 6.8) and runned at 60 V then separated on 12% separating gel (pH 8.8) at 80 V till the dye front reached the bottom of the gel. Finally the gel was stained by using coomassie G250 stain.

Western blotting

Protein estimation of the given sample at 660 nm

§ 100 µg protein will be resolved on apt% of SDS PAGE gel

§ Gel will be pre run, simulataneously gel loading mixtures(sample+dye +DTT) will be prepared and also appropriate pre –stained marker(10µl) will be loaded

Gel will be run at 60V till it crosses stacking and enters resolving gel where voltage will be increased to 80V

§ Gel will be kept in transfer buffer for half an hour at 4ºC after sample has run to sufficient distance on the gel

§ The nitrocellulose membrane was cut according to the size of the gel and was wetted with water to activate it for 1 minute and put in transfer buffer at 4ºC for 20 minutes.

§ The three filter papers were cut according to the size of the gel and membrane, and wetted in anode buffer I and II and cathode buffer respectively

§ The transfer unit was set as filter paper (anode I and II), then membrane ,then gel placed over it,finally filter dipped with cathode buffer.

§ The transfer unit was set at 15V for 1 hour

§ Blocking,1ºAb and 2ºAb incubation will be done

§ Each solution will contain 0.1%Tween 20 to aid in even distribution of solution on the membrane

§ Color will be developed using chemiluminiscent reagents

Data is presented as the relative pixel density of the protein bands normalized to β-actin

RESULT:

Cytotoxic potential of lupeol:

The cytotoxic effect of lupeol on A549 cells was determined with varying concentrations of lupeol (50, 75,100 μM) for 24, 48 and 72 h. The results showed a dose-dependent inhibition of cell proliferation in A549 cells, with the extent of growth inhibition increasing upto 76% as a function of time (24, 48 and 72h) . Based on IC50 (100 μM) value calculated from growth inhibition curve, we selected 100 μM dose of lupeol for 24 h incubation for further studies.

Induction of apoptosis by lupeol:

The decreased viability of A549 cells on treatment with lupeol is associated with induction of apoptosis. Lupeol treatment to A549 cells for 24 h resulted in increased sub G1 peak with related decrease in G1 phase over vehicle treated control. (Fig.1)

	Treatment	% Apoptotic cells
Control	-	0.828±1.21
24 h	50 µM	2.5±1.78
	75 µM	7.36±1.65
	100 µM	10.32±2.31

Levels of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (MMP):

The ROS level and MMP were determined in terms of MFI values. Significant loss of MMP (MFI 75.82) (p < 0.05) was recorded in response to 100 dose of lupeol in comparison to vehicle treated (MFI). While, Lupeol treatment 100 μM for 24 h resulted in a increase in ROS level (MFI 148.23) in comparison to vehicle treated cells (MFI 56.54).

Effects of Lupeol in Expression of Apoptosis Inducing Proteins in A549 Cells

Treatment of lupeol (50-100µM) increases expression of tumor supressor protein p53, pro-apoptotic protein Bax while decreases the expression of anti-apoptotic protein Bcl-

Effects of lupeol in Expression of Mitochondria Mediated Apoptotic Proteins in A549 Cells

Treatment of lupeol (50-100µM) in A549 cells induces release of cyt C protein in cytoplasm due to decrease in mitochondrial membrane potential which leads to cleavage of caspases.

DISCUSSION:

In recent years, considerable efforts have been made to develop chemopreventive agents that would inhibit, retard or reverse the phenomenon of multistage carcinogenesis. Several dietary botanicals have been shown to possess chemotherapeutic and anti-carcinogenic properties due to their anti-inflammatory and anti-oxidant properties. Lupeol is one of the compounds, which has been shown to possess anti-carcinogenic activity in tumor models. Lupeol, present in fruits and medicinal plants, is a biologically active compound that has been shown to have various pharmacological properties in experimental studies. Lupeol and its derivatives are drawing considerable attention for treatment of various types of cancers. Lupeol treatment on cancer cells showed cell growth inhibition, anti-inflammatory effects, and tumor regression. But still very less is known about its mechanism to prevent cancer, as well as its cancer therapeutic activities. Lupeol and its constituents have also been reported to prevent lung cancer progression through modulation in expression of Cox-2 and caspase-3. In addition to the effect of mango extract lupeol, its flavonoids have also been demonstrated to be protective against lung in A549 cell line treated with the tobacco related carcinogens. However, its potential against lung A549 cells have not been studied much. Hence, to understand the anti-carcinogenic mechanism and molecular targets of lupeol in lung carcinogenesis, we selected human epithelial carcinoma A549 cells as an in vitro model to establish a cause and effect relationship. In the present study, we have reported that lupeol can induce apoptosis in A549 cells, through mitochondrial cell death pathway. Our study was designed to define the mechanism(s) of the anti proliferative and apoptotic effects of lupeol in A549 cells. We investigated the role of ROS, caspases and Bcl-2, Bax, cytochrome c protein and mitochondria membrane potential in lupeol-induced apoptosis in A549 cells. Cells were incubated with different concentrations of lupeol then cell morphological changes were observed, DNA damage, cell viability and cell cycle were determined by flow cytometric analysis. Sub-G1 phase was also examined. Western blot analysis was used to determine the levels of Bax and Bcl-2 and apoptosis associated proteins. The results indicated that lupeol induced morphological changes, decreased percentage of viable cells and induced apoptosis dose- and time dependently. The levels of caspase-3, and -9 involved in lupeol-induced apoptosis indicating caspase-dependent pathway was induced by lupeol. We have demonstrated that lupeol-induced cell cycle perturbations of lung cancer A549 cell. Here we showed that treatment of lupeol significantly inhibits the viability of A549 cells suggesting chemotherapeutic effect of lupeol against lung cancer. Here we are reporting that treatment of A549 cells with lupeol for 24 h accumulation of cells in sub G1 phase with increase in appearance of apoptotic cell population. To gain insight into the molecular mechanisms involved in apoptosis caused by lupeol, expression of the apoptosis-related proteins were apprised in A549 cells. Apoptosis has been shown to be triggered by ROS generation. Mitochondria, which play a crucial role in apoptosis, are major sites of ROS generation. Excessive generation of ROS can lead to opening of the mitochondrial permeability transition pore with decline in MMP, which culminate apoptotic cell death. Also it has been suggested that apoptosis is inhibited by Bcl-2, which inactivates Bax through interacting and forming heterodimers. Pro-apoptotic signals induce translocation of Bax specifically to the mitochondria and Bax together with Bak form membrane-integrated homo-oligomers, which permeabilize the outer mitochondrial membrane and trigger a loss of the inner mitochondrial transmembrane potential followed by the release of apoptotic factors like Cytochrome-c from the mitochondria to the cytoplasm. Therefore, the Bcl-2 family proteins have been considered as pivotal players in apoptosis, especially mitochondria-mediated apoptosis. However, in our study on A549 cells, lupeol-induced apoptosis was accompanied by elevation in the Bax to Bcl-2 ratio due to the upregulation of Bax and down regulationof Bcl-2 confirming their essential role for the apoptosis. Bcl-2 family proteins (Bax, Bak, Bcl-2, Bcl-X, etc.,) are known to promote the formation of apoptosome with Apaf1 which in turn activates executioner caspases to orchestrate apoptosis. Here we reported the activation of the caspase 9, caspase 3 by lupeol treatment in A549 cells. The ability of PARP is to repair damaged DNA which is prevented through its cleavage by caspases. Consistent with the above study, here caspases activation leads to PARP degradation upon lupeol treatment. The involvement of a lupeol-induced cleavage of caspases and its effect on apoptosis was further confirmed using the caspase inhibitors in in vitro system. These data suggest that executor caspases including caspase-3 are required in lupeol induced occurrence of apoptosis in A549 cells. It has been previously reported that caspase-3 activation is important for the activation of dietary constituent grape seed proanthocyanidinsinduced proapoptotic pathways in A549 cells. A possible candidate for mediating lupeol-induced inhibition of Akt/PKB signaling is the prosurvival transcription factor NFkB.

CONCLUSION:

The results of the present investigation revealed that lupeol (100µM) treatment showed induction of apoptosis in A549 cells in a dose dependent manner. Cell cycle analysis suggests that lupeol treatment induced sub G1 arrest and triggered ROS generation. A significant loss of mitochondrial membrane potential was recorded in 100 µM dose of lupeol in comparison to vehicle treated. In conclusion, our study exhibit that lupeol has a potent chemotherapeutic effect against human lung cancer cell line (A549) and provide a rationale for future endeavours of lung cancer research.The use of ‘Natural compounds’ have provided opportunity to identify phytochemicals that have health supporting properties without any negative side effects. As a new paradigm in nutrition, perhaps some of these non- essential compounds are proving beneficial to health. An understanding of the basic principles of formulation, processing and new biotechnologies will provide ample opportunity for providing sample foods which will utilize phytochemicals having bioactive components to create products to prevent disease and maintain a healthy life throughout our existence. The future of science and product development in this area will be an exciting adventure for years to come. Trials show several promising, modest, short-term effects of garlic supplements, ginger extracts and many more on lipid and antithrombotic factors. Effects on clinical outcomes are not established, and effects on glucose and blood pressure are none to minimal. High dietary intake of certain phytochemicals may be associated with decreased risks of multiple cancers. There is a large body of evidence that the consumption of fruit and vegetables can decrease the risk of cancer. However, the link between diet and health is extremely complex. Some dietary phytochemicals seem to offer protection in an exposure-related manner and many molecular targets an signaling pathways affected by phytochemicals have been discovered. Although in vitro studies have contributed significantly to our understanding, quite a number use concentrations orders of magnitude greater than those achievable in humans or toxic to normal tissues (exemplified by toxic concentrations of indole-3-carbinol, epigallocatechin-3-gallate, curcumin, lupeol for lung cancer and genistein for breast cells). Such studies may produce results that are physiologically irrelevant, thus hindering predictions of efficacy. Here, we argue for careful consideration to be given to the in vitro experimental conditions under which dietary phytochemicals are investigated. Design features, such as the use of appropriate nontoxic concentrations, extended treatment times, three-dimensional cultures, primary tumor cultures, and comparison of susceptibility of various cancer subtypes, should improve our understanding of their molecular targets. This in turn would facilitate predictions as to their potential usefulness in the clinic.

REFERENCE:

Morgan ME, Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis. 1997; 18:2071–2077.

Nigam N, Prasad S, Shukla Y. Preventive effects of lupeol on DMBA induced DNA alkylation damage in mouse. Food Chem Toxicol 2007; 45:2331-2335.

Prasad S, Nigam N, Kalra N, Shukla Y. Regulation of signaling pathways involved in lupeol induced inhibition of proliferation and induction of apoptosis in human prostatecancer cells. Mol Carcinog 2008; 47:916-924.

Saleem M, Afaq F, Adhami VM, Mukhtar H. Lupeol modulates N Fkappa B and PI3K/Akt pathways and inhibits skin cancer in CD-1 mice. Oncogene 2004; 23:5203-14.

Takiar R, Shobana B. Cancer incidence rates and the problem of denominators a new approach in cancer registries. Asian Pac J Cancer Prev. 2009;10(1):123-6.

Received on 18.10.2014 Modified on 22.10.2014

Res. J. Pharmacology and P’dynamics. 6(4): Oct. - Dec.2014; Page 197-203