Investigating cytotoxicity of plant extracts on MCF-7 cell lines

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INTRODUCTION

Worldwide among leading causes of death in 2012, cancers accounts for 8.2 million deaths and figures as second leading cause of death in the United States (Hoyert et al., 2005). Lung, colorectal, stomach, liver and breast cancers are the most causes of cancerous deaths each year. More than 60% of world’s total new annual cases occur in Africa, Asia and Central and South America. These regions account for 70% of the world’s cancer deaths.It is expected that annual cancer cases will rise from 14 million in 2012 to 22 within the next two decades(Globocan 2012, IARC).

Herbal remedy has been approved worldwide where the plants and plant metabolites are used in the treatment of cancer (Hartwell, 1982). The National Cancer Institute ratified 114,000 anticancer plant extracts from 35,000 plant species from 20 countries (Shoeb, 2005). The isolation of the vinca alkaloids, vinblastine (Balunas) and vincristine (Bertino) from the Madagascar periwinkle, Catharanthus roseus hosted a novel era of the use of plant material as anticancer agents. They were the first agents to advance into clinical use for the treatment of cancer (Cragg and Newman, 2005).

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In the development of formularies and pharmacopoeias, data’s on medicinal plants drive towards a new lane in the discovery of anticancer therapeutic agents. A series of records on plant dependent anticancer drugs from published and unpublished sources was first circulated in December 1967 by Hartwell with monumental information of 3000 species of plants with anticancer property (Graham et al., 2000). After the discovery of plant derived secondary metabolites vinblastine, vincristine, camptothecin derivatives, topotecan, irinotecan and etoposide are examined with much attention in anticancer therapy (Pan et al., 2010 & Indap et al., 2006).

RESULTS AND DISCUSSION

In vitro cytotoxicity of plant extracts on MCF-7 cell lines

Plant flavonoids play a major role in disease preventive and therapeutic properties, henceforth the flavonoid rich plants are explored with great implication in the field of therapy. The consumption of flavonoid rich fruits and vegetables remained to reduce the risk of cancer (Ramos, 2007; Kanadaswami et al., 2005; Ren et al., 2003). Since the phytochemical analysis of S.trilobatum, S.campanulata, S.jambos and T. indica leaf extracts revealed the existence of flavonoids the present cytotoxicity study was executed in accordance with a concept, flavonoids as anticancer agents. The MTT assay was implemented in the present study to rule out the cytotoxic efficacy of selected plant extracts, as the method provides an accurate and reliable quantification of viable cell number in proliferation.

The impact of drug efficacy in therapy always remains instigated from in vitro level before its execution in the in vivo models. The MTT colorimetric assay is an entrenched in vitro method to determine the drug cytotoxicity both in cancer and noncancerous cell lines in terms of cell viability (Abu-Dahab and Afifi, 2007; Mosmann, 1983).

A total of 16 extracts from 4 plant species were screened for their cytotoxic activity against human mammary cancer cells (MCF-7) and the results of cytotoxic assay is depicted in Table 4.6 with their significant IC50 values predicted in Figure 4.9, 4.10, 4.11 and 4.12. It is worth noting that IC50 values ranged between 109- 286 µgmL1, though fairly high, still point subtly towards selective activity. The percentage of antiproliferation was further examined by analysis of variation (ANOVA) compared by Dunnett’s multiple comparison test.

Among four extracts in S.trilobatum the STCLE exhibited 71%, STELE and STMLE showed 57.48% and 53.12% antiproliferation on MCF-7 cells at 300 µgmL1. Our result concurs with the antiproliferative property of S.tilobatum ethanolic leaf extracts on Ehrlich Ascites Carcinoma (EAC) cell line (Xavier et al., 2013) and inhibits mice melanoma and metastasis (Caltagirone et al., 2000), which could be due to the presence of flavonoid quercetin in the plant (Phani et al., 2010). The antiproliferative property of S.trilobatum is confirmative from the American cancer society booms flavonol ‘Quercetin’ plays a vital role in anticancer therapy (Sousa et al., 2007),. Epidemiological studies (Dong and Qin, (2011); Messina et al., 2006) suggest dietary flavonoids may reduce the risk of tumors in colon, breast, prostate, lung and pancreas. A study submits the saponin fraction from S.trilobatum (SFST) has a dose-dependent suppressive effect in cell proliferation (Kanchana and Balakrishnan, 2011). A partially purified Sobatum from S. trilobatum shows cytotoxicity in Dalton's Lymphoma ascites, Ehrlich ascites cell lines and tissue culture cells (L929 and Vero) (Mohanan and Devi, 1997).

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Table 4.6.Percentage cytotoxicity of four plant extracts on MCF- 7 cell lines by MTT assay

Plant Extract

Concentration µg/mL

18.75

37.5

75

150

300

IC50

STALE

0

1.932

3.026

6.889

15.666

ND

STMLE

4.245

14.823

27.108

44.328

53.125

246.288ns

STELE

1.809

11.204

24.356

35.365

57.481

245.523ns

STCLE

4.245

13.445

25.887

41.893

71.051

198.266ns

SCALE

0

0.4861

1.326

9.743

14.306

ND

SCMLE

2.227

9.255

17.936

30.828

41.566

>300

SCELE

5.428

9.673

13.22

29.507

51.635

286.812ns

SCCLE

8.42

15.578

32.15

55.811

67.989

183.19ns

SJALE

0.765

2.384

1.796

14.266

41.935

>300

SJMLE

0.765

7.864

21.388

38.483

69.285

210.491ns

SJELE

0.696

6.681

28.184

56.341

74.809

179.001ns

SJCLE

0.139

25.122

43.215

90.884

99.791

110.819**

TIALE

0

0.3

4.88

5.53

7.81

ND

TIMLE

-0.193

2.962

7.211

14.745

26.142

>300

TIELE

28.074

36.316

46.039

65.872

79.974

191.802**

TICLE

6.761

15.84

25.756

57.694

65.035

109.283ns

Control (DMSO)

0

ND

Note: Values in the parenthesis represents the percentage inhibition of cell proliferation. ND-Not detected; Inhibitory concentration (IC50) with ** represents the values are significantly different at p<0.01, by Dunnett’s multiple comparison test with control (DMSO); ns- no significant difference between the values

The chloroform leaf extracts from S.campanulata (SCCLE) showed 67.98% antiproliferation, whereas the SCELE and SCMLE were effective at 51.6% and 41.5% on MCF-7 cell line at 300 µgmL1. Six common human dietary sources of anticancer flavonols from methanolic leaf extracts of S.campanulata such as kaempferol 3-O-(2-O-ẞ -D-xylopyranosyl)- ẞ -D-galactopyranoside , kaempferol 3-O-(6-O- ẞ -L-rhamnopyranosyl)- ẞ -D-galactopyranoside, acteoside, kaempferol 3-O-(6-O-α-L-rhamnopyranosyl)- α -D-glucopyranoside and quercetin 3-O-(2-O- ẞ -D-xylopyranosyl)- ẞ -D-galactopyranoside has been reported (Yaser and Gouda, 2009). The dietary sources of flavonols in S.campanulata could attribute to the antiproliferation in synergistic or discrete action.

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The chloroform leaf extract of S.jambos (SJCLE) revealed highest cytotoxicity 99.79% (p<0.01) against mammary cancer cell line with an IC50 110.81 µgmL1. Besides the SJELE and SJMLE unveiled 74.8% and 69.28% cytotoxicity with IC50 of 179 and 210 µgmL1 respectively. Two acylated flavonol glycosides 3-O-(4"-O-acetyl)-alpha-L-rhamnopyranoside of mearnsetin (myricetin 4'-methyl ether) and myricetin 3-O-(4"-O-acetyl-2"-O-galloyl)-alpha-L-rhamnopyranoside were reported from the leaves of S.jambos (Mahmoud et al., 2001). Flavonols quercetin, myricitin and myricetin were reported from the leaf extracts of S.jambos (Timbola et al., 2002). Reports suggest the inhibitory property of flavonol quercetin against mice melanoma and invasive metastasis (Sousa et al., 2007, Caltagirone et al., 2000).

At 300 µgmL1 ethanolic leaf extract of T.indica (TIELE) showed 79.97% (p<0.01) and TICLE revealed 65%antiproliferation on MCF-7 cell lines with their IC50 of 191.8 and 109.28 µgmL1 respectively. Effective anticancer activity on acetone and ether extracts of T.indica on BHK-21 cell line was reported (Joshi A, Chauhan, 2013). Tylophorine analogs are a unique class of antitumor compounds that have a mode of action different from known antitumor drugs (Gopalakrishnan et al., 1979). Polar phenanthrene-based tylophorine derivatives (PBTs) N-(2,3- methylenedioxy-6-methoxy-phenanthr-9- ylmethyl)-l-2-piperidinemethanol and N(2,3-methylenedioxy-6-methoxy-phenanthr-9-ylmethyl)-5-aminopentanol showed the highest potency of cytotoxic activity against the A549 human cancer cell line (Gopalakrishnan et al., 1980).

The IC50 concentration of all the active extracts in the study ranged 109.28-286.81 µgmL1 with exception of STALE, SCELE, SCMLE, SJALE, TIALE and TIMLE. The “high” IC50 values are likely may be due to very low concentrations of compounds of interest, which would considerably enrich the bioactivity in the cytotoxic assay.

Morphological observation of MCF-7 cells

With the aid of microscopic technique the process of apoptosis can be perceived and evaluated based on the original morphological criteria (Wyllie et al., 1980), which also determines the structural alterations in cells (Taatjes et al., 2008). As the microscopic examination has been the gold standard for the most precise detection of apoptosis (Yasuhara et al., 2003), microscope was employed in the present study to obtain detailed information about the cell surface of MCF-7 cells.

Normal inverted microscopy was applied to observe the morphological changes in the MCF-7 cells treated and untreated with plant extracts at 72 h post treatment at 400x magnifications. The MCF-7 cell viability results exhibited an apparent decrease of living cells in treated group showing an obvious membrane blebbing, a more prominent growth inhibition and shrinkage of the cells, whereas the untreated cells remained confluent throughout the incubation period. The micrographs of treated cells on acridine orange staining at higher concentration of all the four plant extracts exposed distinctive morphological membrane blebbing, a process associated with apoptosis such as cell shrinkage, chromatin condensation, DNA fragmentation and apoptotic body formation Plates 5(a)–8(d). As a result of chromatin margination and cytoplasm condensation a typical compaction and segregation of the nuclear chromatin was observed in apoptotic micrographs.

The morphological observation also revealed that both extra and intracellular structures were intensely impaired by plant extract treatment. The reduction in number of viable cells after treatment was in accordance with its cytotoxic property exhibited by the plant fractions. Clear signs of apoptosis in our investigation concurs with the study, which determines the progression of condensation in treated cells remains accompanied by nuclear and cell outline convolutions followed by breaking up of the nucleus into discrete fragments to produce membrane bounded apoptotic bodies by budding of the cell (Kerr et al., 1972; Tan et al., 2005). The antiproliferative properties of extracts could be evaluated by counting the number of viable cancer cells, while the apoptogenic properties shall be determined by observing typical morphological changes of apoptosis specifically membrane blebbing. The present study demonstrates that the highest antiproliferative property was exhibited by SJCLE (99.79%) followed by other extracts TIELE (79.97%)>SJELE (74.8%)>STCLE (71%)>SJMLE (69.28) >SCCLE (67.98%)>TICLE (65%)>STELE (57.48%)>STMLE (53.12%) and SCELE (51.63%) via induction of membrane blebbing on MCF-7 cells inducing apoptosis. Apoptosis is well evident in the micrographs of cell lines treated with different extracts in a dose dependent manner of all the four plant extracts.

In general apoptosis is a highly regulated and organized cell death process controlling the development and homeostasis of multicellular organisms that occur under a variety of physiological and pathological conditions. Apoptosis includes cellular morphological change, chromatin condensation and oligonucleosomal DNA cleavage [Sun et al., ]. The cytotoxicity of plant extracts in the present study was exposed to persuade apoptosis and the micrographs demonstrate the morphological characteristics of apoptosis cells containing fragmented nuclei and DNA (Figure 8). The present outcomes indicate clearly that the selected plants, which has been used as a healthcare in traditional medicine acts via programmed cell death. Out of four plants selected the Syzygium jambos and Tylophora indica was found to be potent in antiproliferative activity on MCF-7 cell lines. Additional studies are necessary to determine the molecular mechanisms and its pathways to evaluate potential in vivo anticancer activity of the selected fraction along with its active components.

Natural agents that restrain the proliferation of malignant cells by inducing apoptosis may represent a useful mechanistic approach to both cancer chemoprevention and chemotherapy. There is a growing attention in the use of plant materials for the treatment of various types of cancer and development of safer and more effective therapeutic agents (Lim et al., 2009). Due to additive or synergistic and competing or confounding effects of plant extracts on well-characterized tumor cell lines provide an outline model for the analysis of biological mechanisms produced in clinical effects ((Adams et al., 2006) Salvioli et al ., 2007; Luo and Luo, 2006). Similar kind of studies has aided to identify chemotherapeutic agents that provide selective effects against cancerous cells without untoward effects on normal cells (Ravindranath, 2006).

Importantly, among the 16 extracts tested SJCLE and TIELE exhibited 99.79% (IC50 110.81 µgmL1) and 79.97% (IC50 191.8 µgmL1) reliably significant (p<0.01) antiproliferation respectively. The SJELE and SJCLE displayed antiproliferative effect of 74.8 % and 71 % with IC50 values 179 and 198.26 µgmL1 respectively. A total of 7 extracts such as SCELE, STMLE, STELE, TICLE, SCCLE, SJMLE and STCLE unveiled 51- 70% cytotoxicity against MCF-7 cell line at the highest concentration of 300 μgml1. Three extracts namely STALE, SCALE and TIALE showed least cytotoxicity of 15.6%, 14.3% and 7.81% respectively at 300 µgmL1, conversely the SJALE showed a moderate cytotoxicity 41.93% at 300 µgmL1compared to all other aqueous extracts in the study.

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