Evaluation of cytotoxic activity of protein extracts from the leaves of Morinda pubescens on human cancer cell lines

ABSTRACT

Biologically active proteins isolated from plant species can be used in traditional medicine as prolific resources for new drugs Morinda pubescens Sm., Rubiaceae, is a promising medicinal plant which is widely used in folk medicine to treat fever due to primary complex, ulcer and glandular swellings. In this study, proteins were extracted from the leaves of M. pubescens, and precipitated with ammonium sulphate at various saturation concentrations ranging from 20 to 80%. The precipitated protein sample obtained with 80% saturation was further purified using ultrafiltration membrane (<10 kDa). SDS-PAGE analysis identified the presence of crude and ultrafiltered protein bands. FTIR spectrum of the ultrafiltered protein fractions depicted the presence of hydroxyl and carbonyl groups of proteins. The ultrafiltered proteins exhibited increased cytotoxic activity on A549 cells at the concentrations ranging from 15 to 100 µg/ml. About 98% cell viability was also observed in Vero cells treated with the maximum concentration of 100 µg/ml of ultrafiltered protein extract. DNA fragmentation was observed in A549 cells treated with 10 µg/ml of ultrafiltered proteins, indicating the onset of apoptosis.

Keywords:
Cytotoxic activity; FTIR analysis; Morinda pubescens; MTT assay; Protein extraction

Introduction

Bioactive proteins and peptides from plant sources exhibit different activities, such as antimicrobial, antioxidant, antithrombotic, antihypertensive, hypocholesterolemic, hypoglycemic, immunomodulatory, opioid, and antiproliferative activities. These activities can affect the condition of major body systems, like cardiovascular, digestive, endocrine, immune and nervous system (Ledesma et al., 2009). Plants possessing anticancer activity were found in Violaceae, Rubiaceae, Fabaceae and Cucurbitaceae families (Gerlach and Mondal, 2007). Macrocyclic proteins, such as circulins A (32.8 kDa) and B (31.5 kDa), were isolated from Chassalia parvifolia and cyclopsychotride from Psycho trialongipes which belong to Rubiaceae family. These cyclic proteins show cytotoxic activity, antiHIV and hemolytic activity (Gustafson et al., 1994; Witherup et al., 1994). Anticancerous byproducts are derived from Morinda citrifolia (noni) fruit (A549 human lung carcinoma cells) (Jang, 2012) and b romelain, a protein found in several members of Rubiaceae family are reported to have anti-tumor activity (Marshall and Golden, 2012).

Morinda pubescens Sm., commonly known as Aalis a species of flowering plant of the family Rubiaceae, native to Southern Asia. The bark of M. pubescens, is useful in treating eczema, fever due to primary complex, ulcer and glandular swellings, while leaves are useful for digestive disorders and venereal diseases (Nivas et al., 2011). The preliminary phytochemical study of the methanol extracts of leaf and stem bark of M. pubescens, exhibited antimicrobial and antioxidant properties (Murugan et al., 2012). The aim of this study is to isolate and purify cytotoxic proteins from the leaves of M. pubescens.

Materials and methods

Plant material and reagents

The leaves of Morinda pubescens Sm., Rubiaceae, were collected and authentified (PARC/2012/1384) by Dr. P. Jayaraman, Director of Plant Anatomy Research Centre, Chennai. All buffers and chemicals used were of analytical grade. Human cancer cell line such as A549 (adenocarcinomic human alveolar basal epithelial cells) and Vero cell lines (African green monkey kidney cells) were purchased from NCCS, Pune.

Protein extraction

Leaves of M. pubescens were washed with distilled water and shade dried. The dried leaves were ground to fine powder. About 5 g of powdered leaf sample was extracted with 50 ml of extraction buffer (Ribeiro et al., 2007) consisting of 10 mM Na₂HPO₄, 15 mM NaH₂PO₄, 10 mM KCl, 2 mM EDTA (pH 7.0) and kept in constant stirring for 3 h at 4 ºC. Then the contents were filtered and centrifuged at 5000 × g for 20 min. The crude supernatant was lyophilized and stored for further use. The crude supernatant (lyophilized) was further treated with ammonium sulphate for precipitation of proteins with various saturation limit from 20 to 80%. The concentrations of proteins present in the precipitated samples were estimated by Bradford assay (Bradford, 1976).

Ultrafiltration of protein extracts

The precipitated proteins obtained using 80% saturation of ammonium sulphate from the seeds of M. pubescens were fractionated using ultrafiltration membrane (10 kDa cut-off membrane, Amicon). The concentrated filtered solution containing proteins smaller than the pore size which is less than 10 kDa, was lyophilized and stored at 4 ºC for further use (Ketnawa, 2011).

SDS-PAGE analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a widely used technique to separate proteins according to their electrophoretic mobility (Mahmood et al., 2012). About 40 µl of the crude supernatant was loaded into Lanes 1–4, 7,8 and 40 µl of the ultrafiltered protein sample was loaded into Lanes 9, 10. Lanes 5, 6 represents the protein marker of size ranging from 3.5 kDa to 43 kDa. The gel was kept under electrophoretic run for 2 h at 100 V and the protein bands were identified.

FTIR analysis

Identification of functional groups of the ultrafiltered proteins isolated from the leaves of M. pubescens was performed using Shimadzu Fourier transform infrared spectrophotometer (Surewicz and Mantsch, 1988) Ultrafiltered proteins were homogenized with potassium bromide to obtain a pellet. The pellet was scanned in the infrared absorption region between 400 and 4000 cm^-1 with a resolution of 4 cm^-1 (Widjanarko et al., 2011).

Cytotoxic activity of protein extracts

The ultrafiltered protein fractions were tested for cytotoxic activity against the selected cancer cell lines using MTT assay (Pascariu et al., 2011). Different concentrations of ultrafiltered proteins ranging from 2 to 100 µg/ml were added to each well of 96 well plates. The cells were cultured in 96-well plates (2 × 10⁵ cells per well) in DMEM supplemented with 10% FBS for 24 h. After 24 h the cells were observed under phase contrast microscope and morphology of cells were observed. The medium containing positive control and test samples were removed. MTT (50 µl) dye was added to the wells containing 200 µl of fresh medium. The cell lines were incubated in CO₂ incubator for 4 h. After 4 h of incubation, medium containing dye was removed and 200 µl of DMSO was added to dissolve the formazan crystal. The absorbance was recorded at 570 nm and the percentage of cell viability was calculated using the formula:

where At, absorbance of treated cell; and Ac, absorbance of control (untreated cells).

DNA fragmentation assay

DNA fragmentation analysis (Kalinina et al., 2002) was carried out to evaluate the mechanism of cell death in A549 cancer cell line treated with 10 µg/ml, of ultrafiltered protein extract from the leaves of M. pubescens and incubated at -20 ºC overnight. Cells were freezed and thawed three times for detachment of cells from the flasks. Cells (300 µl) from the flasks was taken in an eppendorf and added with 800 µl of proteinase K buffer was added. About 4 µl of proteinase K was added and kept for 1 h incubation at 56 ºC in a water bath. After incubation, 700 µl of phenol:chloroform:isoamylalchohol (25:24:1) and 100 µl of 5 M sodium acetate were added to the mixture. The mixture was centrifuged for 15 min at 5000 × g, 4 ºC. The supernatant was added with 200 µl of isopropyl alcohol and incubated at -20 ºC for 1 h. After incubation, the contents were centrifuged again at 5000 × g, 4 ºC for 15 min. The supernatant was discarded and 1 ml of 70% ethanol was added to the pellet which was centrifuged for 15 min at 6000 × g, 4 ºC. The supernatant was again discarded and air dried. Nuclease free water (20 µl) was added and stored at -20 ºC. Agarose gel electrophoresis (0.8%) was performed at 100 V.

Statistical analysis

The experiments were carried out in triplicates. The results were calculated as mean along with standard error values. Statistical significance was calculated using one-way analysis of variance (ANOVA). A value of p < 0.05 was considered as statistically significant.

Results and discussion

Plant-based products including proteins and small molecular compounds have been suggested as the favorable drugs for cancer treatment in regard to many adverse effects exerted by current cancer treatments, namely chemotherapy and radiation therapy (Ledesma et al., 2005). The concentration of proteins present in the crude supernatant (1 mg/ml), 80% precipitated sample (0.8 mg/ml) and ultrafiltrate (0.5 mg/ml) were estimated by Bradford assay. Similar studies have been performed by extracting bioactive proteins from various plant sources (Maurya et al., 2011; Kumar and Santhi, 2012). SDS-PAGE gel (12%) showed the presence of protein bands of ultrafiltered protein (lanes: 9, 10) and crude supernatant (lanes: 1–4, 7, 8). Lanes 5, 6 depicts the bands of protein marker ranging from the size of 3.5–43 kDa (Fig. 1).

Fig. 1
SDS-PAGE analysis of protein samples from the leaves of Morinda pubescens Sm.

The FTIR spectrum of the ultrafiltered protein fractions (Fig. 2) detected the peaks at 1631.26 cm^-1 and 3353.97 cm^-1 showing the presence of carbonyl (C=O) and hydroxyl (OH) stretching vibrations. Further FTIR analysis of ultrafiltered protein fractions also depicted IR absorption bands at 2359.95, 2341.43 and 1403.00 cm^-1 indicating the presence of aminoacids, peptides and proteins containing N-H bonds. Characteristic bands found in the infrared spectra of proteins and polypeptides include the Amide I and Amide II. These arise from the amide bonds that link the amino acids. The absorption associated with the Amide I band leads to stretching vibrations of the C=O bond of the amide, absorption associated with the Amide II band leads primarily to bending vibrations of the N-H bond. Since both the C=O and the N-H bonds are involved in the hydrogen bonding that takes place between the different elements of secondary structure, the locations of both the Amide I and Amide II bands are sensitive to the secondary structure content of a protein (Susi and Byler, 1983; Byler and Susi, 1986). Similar studies were done on soya, wheat, corn etc., where protein were isolated which were found out using FTIR studies (Thani et al., 2010).

Fig. 2
FTIR spectral analysis of ultrafiltered protein extract of Morinda pubescens.

The cytotoxic activity of ultrafiltered proteins from leaves of M. pubescens Sm., on normal Vero cells and A549 cancer cells were measured by MTT colorimetric assay. Untreated Vero cell lines were used as control (Fig. 3). Vero cell lines treated with different concentrations of ultrafiltered proteins ranging from 2 to 100 µg/ml, showed increased cell viability of 98% (Figs. 4 and 5). An increased percentage of cell viability of A549 cells was noted at the minimum concentrations of 2–5 µg/ml of ultrafiltered protein extract (Figs. 6–8). Cell viability (50%) of A549 cells treated with 10 µg/ml of ultrafiltered protein extract was also examined (Figs. 6–8). Further it was observed that there was a decreased percentage of cell viability of A549 cells treated with increasing concentrations of ultrafiltered protein extract ranging from 15 to 100 µg/ml (Figs. 6–8). Previous studies were reported on anti-cancer property of proteins extracted from Gynura procumbens, Asteraceae, on breast cancer cell line, MDA-MB-231, at an EC₅₀ value of 3.8 µg/ml (Ng et al., 1992). Similar studies were carried out for the anticancer property of Bidens alba protein-extract against human colorectal cancer (SW 480) cells which depicted marked DNA damages and apoptosis-related cellular morphologies (Hew et al., 2013).

Fig. 3
Photomicrographs (40×) of untreated Vero cells (Control).

Fig. 4
Photomicrographs (40×) of Vero cells treated with different concentrations of ultrafiltered protein extract ranging from 2 to 100 µg/ml, isolated from the leaves of Morinda pubescens.

Fig. 5
Percentage of cell viability of Vero cell lines treated with different concentrations of ultrafiltered protein extract ranging from 2 to 100 µg/ml, isolated from the leaves of Morinda pubescens.

Fig. 6
Photomicrographs (40×) of untreated A549 cells (Control).

Fig. 7
Photomicrographs (40×) of A549 cells treated with different concentrations of ultrafiltered protein extract ranging from 2 to 100 µg/ml, isolated from the leaves of Morinda pubescens.

Fig. 8
Percentage of cell viability of A549 cell lines treated with different concentrations of ultrafiltered protein extract ranging from 2 to 100 µg/ml, isolated from theleaves of Morinda pubescens.

Further DNA fragmentation assay was performed by agarose gel electrophoresis. A DNA ladder characteristic of apoptosis was observed in A549 cells treated with 10 µg/ml of ultrafiltered proteins (Lane 2, Fig. 9). However, the DNA extracted from the untreated Vero cells, appeared as distinct band showing the viability of Vero cells (Lane 4, Fig. 9). Apoptosis, a kind of cellular death is characterized by the early activation of endogenous proteases, cell shrinkage and DNA fragmentation (Nagata, 2000). The nuclear DNA of apoptotic cells shows a characteristic laddering pattern of oligonucleosomal fragments (Nagata et al., 2003). Similar observations were noted with the human colorectal cancer SW480 cells and monocytic leukemia THP-1 cells treated with the protein extracts of Calvatiali lacina, Pleurotus ostreatus and Volvariella volvacea. The viabilities of THP-1 and SW480 cells decreased in a concentration-dependent manner after 24 h of treatment with the protein extracts of the selected plants. Apoptotic analysis revealed that the percentage of SW480 cells in the SubG₁phase (a marker of apoptosis) was increased upon treatment with protein-extracts of Pleurotus ostreatus and Volvariella volvacea, indicating that oligonucleosomal DNA fragmentation existed concomitantly with cellular death (Ong et al., 2008; Jin-Yi et al., 2011).

Fig. 9
Agarose gel depicting DNA fragmentation of A549 cells showing, Lane1: Marker (M) – 1 kbp ladder; Lane 2: A549 cells treated with 10 µg/ml of ultrafiltered protein extract; Lane 4: Untreated Vero cell lines.

In conclusion the ultrafiltered proteins extracted from the leaves of M. pubescens exhibited significant cytotoxic activity on A549 cells at the IC₅₀ concentration of 10 µg/ml. Besides, the ultrafiltered protein fraction did not show any inhibitory effects on the proliferation of Vero cells. Further, DNA fragmentation was observed in A549 cells treated with ultrafiltered proteins, thereby indicating the onset of apoptotic cell death. Thus, the results obtained in this study suggest that the ultrafiltered proteins (10 kDa) with apoptosis-inducing activity, isolated from the leaves of M. pubescens can act as potential anticancer agents in cancer chemotherapy.

• Ethical disclosures
  Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.
  Confidentiality of data. The authors declare that no patient data appear in this article.
  Right to privacy and informed consent. The authors declare that no patient data appear in this article.

References

• Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
• Byler, D.M., Susi, H., 1986. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymer 25, 469-487.
• Gerlach, S.L., Mondal, D., 2007. The bountiful biological activities of cyclotides. CYS 3, 169-177.
• Gustafson, K.R., Sowder, R.C., Henderson, L.E., Parsons, I.C., Kashman, Y., Cardellina, J.H., McMahon, J.B., Buckheit, R.W., Pannell, L.K., Boyd, M.R., 1994. Circulins A and B: novel HIV inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia K. Schum. J. Am. Chem. Soc. 116, 9337-9338.
• Hew, C., Khoo, B.Y., Gam, L.H., 2013. The anti-cancer property of proteins extracted from Gynura procumbens (Lour.) Merr. PLOS ONE, http://dx.doi.org/10.1371/journal.pone.0068524
  » http://dx.doi.org/10.1371/journal.pone.0068524
• Jang, B.C., 2012. The fruit juice of Morinda citrifolia L. (noni) downregulates hif-1α protein expression through inhibition of pkb, erk-1/2, jnk-1 and s6 in manganese-stimulated A549 human lung cancer cells. Int. J. Mol. Med. 29, 499-504.
• Jin-Yi, W., Chi-Hung, C., Wen-Huei, C., King-Thom, C., Yi-Wen, L., Fung-Jou, L., Ching-Hsein, C., 2011. Anti-cancer effects of protein extracts from Calvatiali lacina, Pleurotus ostreatus and Volvariella volvacea Evid. Based Complement. Altern. Med., http://dx.doi.org/10.1093/ecam/neq057
  » http://dx.doi.org/10.1093/ecam/neq057
• Kalinina, T.S., Bannova, A.V., Dygalo, N.N., 2002. Quantitative evaluation of DNA fragmentation. Bull. Exp. Biol. Med. 134, 554-556.
• Ketnawa, S., 2011. Extraction of bromelain from pineapple peels. Food Sci. Technol. Int. 17, 395-402.
• Kumar, D.J., Santhi, R.J., 2012. Antioxidant and cytotoxic effects of hexane extract of Morinda pubescens Sm. leaves in human liver cancer cell line. Asian Pac. J. Trop. Med. 5, 62-66.
• Ledesma, H.C., Hsieh, C., de Lumen, B.O., 2005. Chemopreventive properties of peptide lunasin: a review. Protein Pept. Lett. 20, 424-432.
• Ledesma, H.B., Hsieh, C., De Lumen, B.O., 2009. Lunasin, a novel seed peptide for cancer prevention. Peptides 30, 426-430.
• Mahmood, A., Raja, G.K., Mahmood, T., Gulfraz, M., Khanum, A., 2012. Isolation and characterization of antimicrobial activity conferring component(s) from seeds of bitter gourd (Momordica charantia L.). J. Med. Plants Res. 6, 566-573.
• Marshall, S.J., Golden, K.D., 2012. Characterization of bromelain from Morinda citrifolia L., (noni). JSR 4, 445-456.
• Maurya, D.K., Nandakumar, N., Devasagayam, T.P.A., 2011. Anticancer property of gallic acid in A549, a human lung adenocarcinoma cell line, and possible mechanisms. J. Clin. Biochem. Nutr. 48, 85-90.
• Murugan, M., Mohan, V.R., Thamodharan, V., 2012. Phytochemical screening and antibacterial activity of Gymnema sylvestre R.Br., and Morinda pubescens Sm. JAPS 2, 73-76.
• Nagata, S., 2000. Apoptotic DNA fragmentation. Exp. Cell Res. 256, 12-18.
• Nagata, S., Nagase, H., Kawane, K., Mukae, N., Fukuyama, H., 2003. Degradation of chromosomal DNA during apoptosis. Cell Death Differ. 1, 108-116.
• Ng, T.B., Chan, W.Y., Yeung, H.W., 1992. Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-aids activities from Cucurbitaceae plants. Gen. Pharmacol. 23, 579-590.
• Nivas, D., Gaikwad, D.K., Chavan, P.D., 2011. Antioxidant potential of Morinda pubescens Sm. fruits. J. Pharm. Res. 4, 829-831.
• Ong, P.L., Weng, B.C., Lu, F.J., Lin, M.L., Chang, T.T., Hung, R.P., Chen, C.H., 2008. The anticancer effect of protein-extract from Bidens alba in human colorectal carcinoma SW480 cells via the reactive oxidative species- and glutathione depletion-dependent apoptosis. Food Chem. Toxicol. 46, 1535-1547.
• Pascariu, M., Nevoie, A., Jitaru, D., Carasevici, E., Luchian, T., 2011. The evaluation of biological effect of cytotoxic peptides on tumor cell lines. Dig. J. Nanomater. Biostruct. 7, 79-84.
• Ribeiro, S.F.F., Carvalho, André O., Cunha, M., Rodrigues, R., Cruz, L.P., Melo, V.M.M., Vasconcelos, I.M., Melo, E.J.T., Gomes, V.M., 2007. Isolation and characterization of novel peptides from chilli pepper seeds. Toxicon 50, 600-611.
• Surewicz, W.K., Mantsch, H.H., 1988. New insight into protein secondary structure from resolution-enhanced infrared spectra. Biochim. Biophys. Acta 952, 115-130.
• Susi, H., Byler, D.M., 1983. Protein structure by Fourier transform infrared spectroscopy: second derivative spectra. Biochem. Biophys. Res. Commum. 115, 391-397.
• Thani, W., Vallisuta, O., Siripong, P., Ruangwises, N., 2010. Anti-proliferative and antioxidative activities of Thai noni/Yor (Morinda citrifolia L.). Southeast Asian J. Trop. Med. Public Health 41, 482-489.
• Widjanarko, S.B., Nugroho, A., Estiasih, T., 2011. Functional interaction components of protein isolate and glucomannan in food bars by FTIR and SEM studies. Afr. J. Food Sci. 5, 12-21.
• Witherup, K.M., Bogusky, M.J., Anderson, P.S., Ramjit, H., Ransom, R.W., Wood, T., Sardana, M., 1994. Cyclopsychotride A, a biologically active, 31-residue cyclic peptide isolated from Psychotria longipes J. Nat. Prod. 57, 1619-1625.

Publication Dates

• Publication in this collection
  Jan-Feb 2017

History

• Received
  10 Sept 2015
• Accepted
  08 Aug 2016