Introduction

The total number of new cancer cases is estimated to be 15 million and the number of cancer deaths will be approximately 12 percent of world population. Breast cancer is the second leading cause of cancer death in females (Bray et al., 2018). The chemotherapeutic drugs have side-effects and sometimes create a significant problem in the treatment of cancer. Use of plant-derived compounds for cancer therapy, may reduce adverse side effects (Desai et al., 2008). Compounds derived from plants have properties to inhibit proliferation of cancer cells and to induce apoptotic cell death, thus they exhibit anticancer activity (Greenwell & Rahman, 2015). Bioactive compounds derived from plants show anti-cancer activities. I n vitro and in vivo studies of triterpenoids belongs to the lupane, oleanane, ursane, and cucurbitacin groups for chemo prevention and therapy of breast cancer, and pancreatic cancer, showed that they possess pleiotropic mode of effects for cancer (Wang et al., 2012). Cissus quadrangularis, a perennial plant of Vitaceae family is safe and free of adverse effects at the commonly used dosage level (Jstohs & Ray, 2013)and helps in pain reduction (Brahmkshatriya, Shah, Ananthkumar, & Brahmkshatriya, 2015), treatment of arthritis and other inflammatory disorders (Kumar, Gupta, Singh, & Arunraja, 2015). Cissus quadrangularis selectively induces cytotoxicity, ROS liberation, G1 phase cell cycle arrests only in HeLa cancer cells without affecting the normal skin cells and inhibits the growth of tumoroid (Sabasheikha et al., 2015). Cissus quadrangularis have shown anticancer activity in HeLa cell line, MCF-7 cell line, KB oral epidermoid carcinoma cell line, A431 cell line and EAC (Ehrlich Ascites Carcinoma) Cell line (Rajamaheswari, Visveswaran, Muthukumar, Murugesan, & Banumathi, 2017). The ethanol and methanol extracts of C. quadrangularis shows anticancer activity with the IC₅₀ value at the concentration of 62.5µg/ml and 125µg/ml for HeLa and Vero cell line respectively (Dwivedi & Seethalakshmi, 2013). Thus, C.quadrangulris seems to possess promising anticancer properties. The objective of this article is to study the anti-cancer and anti-metastatic activities of the crude extract of the plant in MCF-7 cell line.

Materials and Methods

Reagents and Chemicals

Phosphate-Buffer Saline, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) Reagent, DMSO, Annexin Binding buffer, PI working solution, FITC, Digestion buffer, Phenol, chloroform, isoamyl alcohol, sodium acetate, ethyl alcohol, Tris-EDTA buffer, Bradfords Reagent, Coomassie blue, Blocking buffer, PBS Tween 20, Primary Antibody against Caspase 7/9 and Secondary Antibody, Chromogen O Dianisidine and 5N HCl.

Cell Culture

MCF-7cells were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% FBS, Penicillin (100U/ml), Streptomycin (100µg/ml) and Amphoteracin B (2.5µg/ml). Cultured cell lines were kept at 37ºC in a humidified 5% CO₂ incubator (NBS Eppendorf, Germany).Two days old confluent monolayer of cells were trypsinized and the cells were suspended in 10% growth medium, 100µl cell suspension (5x10⁴ cells/well) was seeded in 96 well tissue culture plate and incubated at 37ºC in a humidified 5% CO₂ incubator.

Sample Preparation

The dried Cissus quandrangularis plant added to 200ml of 70% ethanol and mixed thoroughly in rotatory shaker for 48 hrs. Then the extract is obtained by squeezing the mixture using muslin cloth. Ethanol is evaporated by drying the extract. The dried extract was transferred to the centrifuge tube to which 30ml of distilled water was added. Fractionation was done with a separating funnel. To the funnel, the homogenized mixture and 100ml of 90% ethyl acetate were added. After thorough shaking, it was left to settle down. The supernatant was transferred to a petri dish and left for drying. To the organic phase, 30 ml of 90% ethyl acetate was added and left for separation. The final dried extract of carica papaya was scrapped off and transferred to an Eppendorf tube. Then it was weighed and found to be 95mg. 1 mg of sample was weighed and dissolved in 1 mL DMEM using a cyclomixer. The sample solution was filtered through 0.22 µm Millipore syringe filter to ensure the sterility.

MTT Assay

For cell viability assays, MCF-7 breast cancer cells were grown in Dulbecco's modified Eagle's medium (DMEM) with C.quadrangularis extract and were added to the wells. After 24 hours of incubation period, the samples were removed and 30µl of reconstituted MTT solution was added to all test and control wells, the plate was gently shaken, then incubated at 37ºC in a humidified 5% CO₂ incubator for 4 hours. After the incubation period, the supernatant was removed and 100µl of MTT Solubilization Solution (Dimethyl sulphoxide, Sigma Aldrich, USA) was added and the wells were mixed gently by pipetting up and down in order to solubilize the formazan crystals. The absorbance was measured by using microplate reader at a wavelength of 540 nm (Vijayalakshmi, Kumar, Priyadarsini, & Meenaxshi, 2013).

Wound Healing Assay

The cells were trypsinized and seeded at a density of 200,000 cells per well into 12-well plate for 24 h incubation (∼90% confluence). Scratch wounds were made by a sterile 1 mL pipette tip through a pre-marked line. After removal of the resulting debris from five lineal scratches, the cell monolayer was subsequently rinsed three times with PBS, followed by incubation with C.quadrangularis sample of concentration 141.369µg/mL for 0 hour, 24 hours, 48 hours and 72 hours (Zhu et al., 2018). The wound areas were displayed by taking images just above the interchanges between scratched wound areas and pre-marked lines. The effect of the sample on wound closure was determined microscopically (4X magnification, Olympus CKX41) after incubation and was measured in terms of area using MRI-Image J analysis software.

Caspase 7/9 Induction Detection Assay

After attaining sufficient confluency, cells were exposed to the IC₅₀ concentration of the C.quadrangularis extract (141.369µg/mL) and incubated for 24 hours at 37ºC in a humidified 5% CO₂ incubator. For control, nontreated cells were also maintained. After incubation, the supernatant was collected and 100μL of it was added to the 96 well plate and kept for incubation at 37⁰C overnight. The following day, the supernatant was taken for protein estimation and the wells were drained and washed with PBS twice. 200μl of freshly prepared blocking buffer in PBS was added and incubated for 1 hour at room temperature. It was then washed twice with PBS TWEEN. 100μl of primary antibodies(Caspase 7 and Caspase 9) each was added and left for 2 hours at room temperature. After incubation it was washed with PBS TWEEN two times. 100µL of secondary antibody (HRP Conjugate, Santacruez, USA) was added and left for 1 hour at room temperature. It was then washed with PBS TWEEN for two times. 200µL of O- dianizdine hydrochloride (Sigma Aldrich,USA) was added and left for 30 minutes at room temperature. The reaction was stopped by adding 5N HCl (50μl).OD was read at 415 nm in an ELISA reader (Nassar, Aisha, Suede, & Majid, 2012).

Annexin V FITC Assay

The confluent MCF-7 cells were treated with the IC50 concentration of C.quadrangularis sample (141.369µg/mL) and incubated for 24 hours. The cells were trypsin zed after incubation and 100μL of the cells in suspension was transferred to a separate tube. To the tube, 100μL of the Muse™ Annexin V & Dead Cell Reagent were added. The tube was mixed thoroughly by pipetting up and down or vortexing at a medium speed for 3 to 5 seconds followed by incubation for 20 minutes at room temperature in the dark. The cells were analyzed in a flow cytometer using Muse flow cytometry software. Cells were gated against untreated control cells and analyzed for apoptosis using Muse FCS 3.0 software (Kntayya, Ibrahim, Ain, Iori, & Ioannides, 2018).

Clonogenicity Assay

The cells were exposed with C.quadrangularis sample of 141.369µg/mL and control cells were incubated for 24 hours at 37ºC in a humidified 5% CO₂ incubator. The clono genic assay was performed in a 6 well plate. 1% agarose and 0.8% agarose was prepared and kept in 56⁰C water bath. The bottom layer was made by adding 2 ml of 1% agarose mixed equally with DMEM. It was then kept undisturbed for few minutes until it solidifies. 5ml of 0.8% agarose was mixed with 6 ml of DMEM. The top layer was made by adding 2ml of the above mixture. It was then kept for15 minutes. The transfected cells were trypsinized and spread carefully over the top layer. Dishes were incubated to allow colony formation for 14 days. The colonies were first fixed with formaldehyde (4%) for 2 hours and then stained with 0.005% Crystal violet for 30 minutes. Each assay was made in triplicate and only colonies containing at least 50 cells were counted (Kumar, Sharma, Yadav, & Dey, 2017).

DNA Fragmentation Assay

The cells treated with C.quadrangularis sample of IC₅₀ concentration of 141.369µg/mL was incubated for 36 hours. Then the cells were trypsinised and centrifuged for 5 minutes at 500 x g at 4⁰C to form pellets.The cells were washed with ice cold PBS and re-pelletized. The pellet was then resuspended in 0.3mL of Digestion Buffer. The sample was incubated at 50⁰C for 12 hours. It was then extracted with phenol/ chloroform/ iso amyl alcohol (IAA) in the ratio 25:24:1followed by centrifugation for 10 minutes at 1700 x g.Aqueous layer was transferred to a new tube and was added with half volume of 7.5M ammonium acetate and 2 volumes of 100% ethanol. DNA was pelletized by spinning for 2minutes at 1700 x g. It was then washed with 70% ethanol. Ethanol was decanted and air dried. DNA was resuspended in1mg/ml TE buffer (Kumar et al., 2017; Rahbarsaadat, Saeidi, Vahed, Barzegari, & Barar, 2017).

Results and Discussion

MTT Assay

MTT assay, only active cells form formazan product (purple colour), the viability of the cells can be measured easily. Then, it was observed that the number of cells decreased with increased dosage. The cells treated with C.quadrangularis showed lesser viability. The percentage of the cells that were viable were determined by calculating the optical density of the cells treated with the extracts against blank as control containing only the cells, at 540nm. The percentage of cell viability in C.quadrangularis extract is 94.98%, 90.58%, 85.91%, 78.97%, 63.65% for the concentrations 6.25, 12.5, 25, 50, 100 respectively. The inhibitory concentration, IC₅₀ value of C.quadrangularis was found to be 141.369µg/mL, using ED50PLUS V1.0.

Wound Healing Assay

The wound closure at every interval was compared with the initial wound width (at 0hrs). The percentage wound area in the cells treated with C.quadrangularis was 55.86% at 24hrs, 54.75% at 42hrs and 47.55% at 72hrs. The results as shown in Figure 1 demonstrates that in the control, the wound closure was almost complete, after 48hrs incubation and the wound closure was minimum in the cell groups treated with the extract which shows that there is inhibition to the cell migration in a time-dependent manner.

Caspase 7/9 Induction Detection Assay

In this study, the effect of C.quadrangularis on caspase 7/9 activation was analysed. The Caspase 7 activity in control is 0.1356 unit/mg of protein and in the cells treated with the C.quadrangularis extract is 0.2603 activity unit/mg of protein. The Caspase 9 activity in the control is 0.1273 activity unit/mg of protein and in the cells treated with C.quadrangularis is 0.3110 activity unit/mg of protein. The result shows that Caspase activity was enhanced while the MCF-7 cells were treated with samples.

Annexin V FITC Assay

It can be observed that after staining the cells with FITC annexin V and PI, the apoptotic cells showed green fluorescence, dead cells showed red and green fluorescence and live cells showed little or no fluorescence in both control and C.quadrangularis treated cells, shown in Figure 2. These populations were distinguished using a flow cytometer with the 488 nm line of an argon-ion laser for excitation. Using Muse FCS 3.0 software, the percent of apoptotic cells in the control was found to be 8.18% and in cells treated with C.quadrangularis was found as 35%.

The percentage of cell distribution in control cells under live state, early apoptotic, late apoptotic/dead, debris, total apoptotic are 91.74%, 2.96%, 5.22%, 0.08%, 8.18% respectively.

The percentage of cell distribution in cells treated with sample – C.quadrangularis under live state, early apoptotic, late apoptotic/dead, debris, total apoptotic are 63.12%, 14.30%, 20.70%, 1.88%, 35% respectively.

Clonogenicity Assay

The colonies containing at least 50 cells were considered and the total number of colonies were counted as 381 in control and 107 in the cells treated with the C.quadrangularis extract and the colony formation is shown in Figure 3.

DNA Fragmentation Assay

DNA fragmentation assay is performed to study the apoptotic activity in the cell group treated with C.quadrangularis extract. As a result of this assay, a DNA laddering pattern as shown in Figure 4, can be observed after running in agarose gel electrophoresis. On comparison with the control, the DNA laddering pattern of the C.quadrangularis treated cells shows more apoptotic cell death.

Regulation of growth is lost in tumor cells and they survive beyond their normal lifespan and proliferate abnormally. Anticancer agents derived from plants can block proliferation, resulting in cell cycle arrest and apoptosis (Feitelson et al., 2015). MTT assay is a standard colorimetric assay that measures the cell respiration and the amount of formazan produced to give the number of viable cells in the culture treated with the desired compound. The more the number of viable cells in the culture, the more formazan crystal formation, from which the cell proliferation occurs and thus the cytotoxicity of the sample is determined. IC₅₀ is the concentration of sample that causes 50% cell death and it shows the amount of cytotoxicity (Florento, Matias, Tuano, Santiago, & Cruz, 2012). MCF-7 cells treated with ethyl acetate fraction of ginger leaf extract of concentration 100µg/mL, shows cell viability reduced to 36%, after 24h (Park et al., 2014). Ethanolic extract of Arbuscular mycorrhizal fungi (AMF) induced ginger shows IC₅₀ concentration of 12.5µg/mL on T47D cell line (Suharty, Wahyuni, & Dachriyanus, 2018). IC₅₀ concentration of 1319.25µg/mL and 111.36µg/mL are determined in the test on MCF-7 cell line treated with serial dilution of 3000µg/mL aqueous papaya leaf extract and 100µg/mL of Quercetin, respectively (Zuhrotun, Astuti, Murdiati, & Mubarika, 2017). IC₅₀ values of caffeic acid (CA) and caffeic acid phenethyl ester (CAPE) in MDA-MB-231 for 24 hours using SRB method, were 139.80 µg/mL and 60.86 µg/mL, respectively (Kabała-Dzik et al., 2017). In this study, the IC₅₀ value of ethyl acetate extraction of Cissus quadrangularisis found to be 141.369 µg/mL which is more than that of ginger and caffeic acid phenethyl ester and thus C.quadrangularis is not so effective than these. But the IC50 value of C.quadrangularis is nearer to that of Quercetin and caffeic acid, hence they are slightly equal in potential.

Metastasis initially begins with the local invasion of tumor into the surrounding host tissue. Tumor cells migrate from their confined primary site, singly or co-ordinately for facilitating the invasion (Scully & Bay, 2012). Thus inhibition of migration will arrest metastasis. Effect of Cissus quadrangularis on cellular motility and migration is studied by wound healing assay, since they are the primary need of tumor invasion, intravasation, diffusion to distant site, extravasation and colonization at secondary site.100 µM of caffeic acid promoted migration inhibition of the MDA-MB-231 cells with the wound area value 49% after 8 h, after 16 h, and 9% after 24 h. 100 µM of Caffeic acid phenethyl ester stopped the MDA-MB-231 cells from migrating at 75%, after 8 h, 72% after 16 h, 68% after 24 h. 100µM of Calycosin after 48 hours of treatment reduced the migration of MCF-7 cells by 57.6% and reduction of migration in T47D cells by 63% (Li et al., 2017). The result of wound healing assay indicates there is significant inhibition of migration when the cells are treated with cissus quadrangularis at 24, 48 and 72hrs. The wound closure was minimum in the cell groups treated with the sample. Hence, there is inhibition to the cell migration in a time-dependent manner.

The caspase7 activity was increased to two folds when the MCF-7 cell line was treated with the sample C.quadrangularis. The untreated sample has a caspase7 activity of about 0.135651 activity units/mg of protein. The caspase9 activity was increased to two folds when the MCF-7 cell line was treated with the sample C.quadrangularis. The untreated sample has a caspase9 activity has decreased folds. Caspase pathways are considered as suitable candidate targets for anticancer drug discovery. It has been reported that some commonly used plant-derived anticancer drugs such as tamoxifen, doxorubicin, etoposide and cisplatin promote apoptosis in tumor cells through caspase pathways.

The induction of apoptosis by the C.quadrangularis ethyl acetate extract in the MCF-7 cell lines was confirmed using Annexin V assay with flow cytometric analysis. Annexin V, having a strong affinity for PS is used as a probe for detecting apoptosis (Rebecca & Wong, 2011).

Further, on comparison with other plant extracts such as, black turtle bean (BTB) for which, the total percentage of apoptotic cells was 94.47% (Lakshmanan & Batra, 2013) for Alpiniaofficinarum (belonging to ginger family), total percentage of apoptotic cells was 77.1% (Ghil, 2013; Kumar et al., 2017), it is observed that, even though the result is not comparably best, Cissus quadrangularis (with total apoptotic cells at 35%) does possess good apoptotic activity, thereby contributing to its anti-cancerous property.

In clonogenicity assay, cells are grown in between two layers of soft agar with the layer at the bottom containing a higher concentration of agar than the other. This does not allow the cells to attach to the culture plate but transformed and carcinogenic cells are capable of forming colonies (Taddei, Giannoni, Fiaschi, & Chiarugi, 2012). The number of colonies counted in control (381 colonies) clearly demonstrates that there is a very high possibility of metastatic activity. Whereas, the number of colonies counted in the cells treated with C.quadrangularis extract was greatly reduced to 107. When comparing with plant extracts such as Pelingo apple juice which showed formation of a little more than 20 colonies (Borowicz et al., 2014) and for Gynuramedica(of concentration 40µM), the number of colonies formed were a little over 100 (Schiavano et al., 2015), C.quadrangularis inhibits the formation of colonies which reduces the probability of metastatic activity of the carcinogenic cells.

Apoptosis is characterised by chromatin condensation and fragmentation, overall cell shrinkage/rounding and formation of apoptotic cell bodies and any disturbance to the regulation of apoptosis plays a major role in cancer growth. Thus, any compound which regulates apoptosis is considered anti-cancerous. DNA fragmentation is the hallmark of apoptosis and DNA ladder observation is one of the methods to assess it (Yi et al., 2016). DNA laddering pattern in agarose gel electrophoresis confirms DNA cleavage in the cells as result of apoptosis induction.

Conclusion

It can be concluded that Cissus quadrangularis has potential anti-cancer and anti-metastatic activity compared to other medicinal plants. The phytomedicinal properties were identified by the anti-cancerous activity through MTT assay, DNA fragmentation assay, caspase 7/9 induction detection assay and Annexin-V FITC assay and the anti-metastatic activity through wound healing assay and clonogenicity assay.