International Journal of
Molecular Sciences Article
Ficus umbellata Vahl. (Moraceae) Stem Bark Extracts Exert Antitumor Activities In Vitro and In Vivo Kevine Kamga Silihe 1 , Stéphane Zingue 2,3,4,5 , Evelyn Winter 4 , Charline Florence Awounfack 3 , Anupam Bishayee 6, *, Nishil N. Desai 6 , Leônidas João Mello Jr. 4 , Thomas Michel 7 , Francine Nzufo Tankeu 1 , Derek Tantoh Ndinteh 5 , Sara Honorine Riwom 1 , Dieudonné Njamen 3,5 and Tânia Beatriz Creczynski-Pasa 4, * 1 2 3 4 5 6 7
*
Department of Biochemistry, Faculty of Sciences, University of Yaounde 1, Yaounde 812, Cameroon; [email protected] (K.K.S.); [email protected] (F.N.T.); [email protected] (S.H.R.) Department of Life and Earth Sciences, Higher Teachers’ Training College, University of Maroua, Maroua 55, Cameroon; [email protected] Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé 1, Yaounde 812, Cameroon; [email protected] (C.F.A.); [email protected] (D.N.) Department of Pharmaceutical Sciences and Department of Biochemistry, Federal University of Santa Catarina, Florianópolis 88040-900, Brazil; [email protected] (E.W.); [email protected] (L.J.M.) Department of Applied Chemistry, Faculty of Sciences, University of Johannesburg, Doornfontein 2028, South Africa; [email protected] Department of Pharmaceutical Sciences, College of Pharmacy, Larkin University, Miami, FL 33169, USA; [email protected] Institute of Chemistry of Nice, Faculty of Sciences, University Côte d’Azur, Nice F-06108 Nice CEDEX 2, France; [email protected] Correspondence: [email protected] or [email protected] (A.B.); [email protected] (T.B.C.-P.); Tel.: +1-305-760-7511 (A.B.)
Academic Editor: Leticia M. Estevinho Received: 29 March 2017; Accepted: 3 May 2017; Published: 23 May 2017
Abstract: A Ficus umbellata is used to treat cancer. The present work was therefore designed to assess antitumor potentials of F. umbellata extracts in nine different cell lines. Cell cycle, apoptosis, cell migration/invasion, levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), caspases activities as well as Bcl-2 and Bcl-xL protein content were assessed in MDA-MB-231 cells. The 7,12-dimethylbenz(a)anthracene (DMBA)-induced carcinogenesis in rats were also used to investigate antitumor potential of F. umbellata extracts. The F. umbellata methanol extract exhibited a CC50 of 180 µg/mL in MDA-MB-231 cells after 24 h. It induced apoptosis in MCF-7 and MDAMB-231 cells, while it did not alter their cell cycle phases. Further, it induced a decrease in MMP, an increase in ROS levels and caspases activities as well as a downregulation in Bcl-2 and Bcl-xL protein contents in MDA-MB-231 cells. In vivo, F. umbellata aqueous (200 mg/kg) and methanol (50 mg/kg) extracts significantly (p < 0.001) reduced ovarian tumor incidence (10%), total tumor burden (58% and 46%, respectively), average tumor weight (57.8% and 45.6%, respectively) as compared to DMBA control group. These results suggest antitumor potential of F. umbellata constituents possibly due to apoptosis induction mediated through ROS-dependent mitochondrial pathway. Keywords: Ficus umbellata; apoptosis; estrogen-dependent tumors; DMBA; cell invasion
1. Introduction Cancer is a group of diseases characterized by the unregulated proliferation of abnormal cells that invade and disrupt surrounding tissues [1]. It is a source of significant morbidity worldwide, responsible for over 8.2 million deaths per year and a serious public health problem in both developed Int. J. Mol. Sci. 2017, 18, 1073; doi:10.3390/ijms18061073
www.mdpi.com/journal/ijms
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responsible for over 8.2 million deaths per year and a serious public health problem in both developed and developing countries [2]. and Breast and gynecological cancers (e.g., ovarian and uterus) and developing countries [2]. Breast gynecological cancers (e.g., ovarian and uterus) are the aremajor the major cause of cancer death in women of developing countries, and represent 19% cancers cause of cancer death in women of developing countries, and represent 19%ofof cancers worldwide World Health Organization, the incidence of breast and worldwide[3]. [3]. According According totothethe World Health Organization, the incidence of breast and gynecological gynecological cancers is predicted to increase in the future and 70% new cases will be observed in cancers is predicted to increase in the future and 70% new cases will be observed in developing developing countries [2]. In Africa, the most common cancers in women are breast and cervical ones countries [2]. In Africa, the most common cancers in women are breast and cervical ones [4]. However, [4].endometrial However, endometrial ovarian cancers are also routinely incidence of ovarian and ovarian and cancers are also routinely observed. Theobserved. incidenceThe of ovarian cancer increases cancer increases worldwide by about 10-fold in women during the perito post-menopausal period, worldwide by about 10-fold in women during the peri- to post-menopausal period, when compared when compared to premenopausal women With per 3500year, deaths year, cancer the ovarian is the of to premenopausal women [5]. With 3500[5]. deaths the per ovarian is thecancer most lethal most lethal of gynecologic malignancies and a relatively uncommon cancer, making testing of drugs gynecologic malignancies and a relatively uncommon cancer, making testing of drugs for primary forprevention primary prevention difficult [6,7]. difficult [6,7]. Nowadays, regimensagainst against breast and gynecological cancersradiotherapy, include Nowadays,therapeutic therapeutic regimens breast and gynecological cancers include radiotherapy, chemotherapy, immunotherapy and hormonal therapies, and, in advanced stages of chemotherapy, immunotherapy and hormonal therapies, and, in advanced stages of the disease, surgery theintervention disease, surgery intervention can be required [8,9]. Despite substantial improvements in can be required [8,9]. Despite substantial improvements in survival, the management survival, theis management is limited to its high scarcity of diagnosis of cancer limited due toofitscancer high cost, scarcity due of diagnosis and cost, treatment, limited access to and health treatment, limited access to health services found in African countries [10], and most importantly the as services found in African countries [10], and most importantly the problem of resistance as well problem of resistance as well as high systemic toxicity ofdrugs available drugs [11]. The is high systemic toxicity of available chemotherapeutic [11].chemotherapeutic The need for primary prevention need for primary prevention is worthwhile and novel efficacious, non-toxic and cost-effective worthwhile and novel efficacious, non-toxic and cost-effective therapeutic agents should be developed. therapeutic should be and developed. Moreover, epidemiological and laboratory studies have Moreover,agents epidemiological laboratory studies have provided evidence that naturally occurring provided that naturally occurring dietary or plantagainst components may exert[12–14]. protective effects dietary evidence or plant components may exert protective effects various cancers Increasingly, against various cancers [12–14]. Increasingly, more medicinal plants are being screened for more medicinal plants are being screened for anticancer properties and may provide effective anticancer properties and may provide effective therapeutic agents. therapeutic agents. The primary prevention of of breast and gynecological cancers remains an an important issue forfor The primary prevention breast and gynecological cancers remains important issue developing countries, where 80% of the population still resorts to traditional medicine for their developing countries, where 80% of the population still resorts to traditional medicine for their primary primary care In thisFicus scope, Ficus umbellata, a plant in subtropical regions,up growing health health care [15]. In[15]. this scope, umbellata, a plant found in found subtropical regions, growing to 6–10 m uphas to 6–10 has been investigated. F. umbellata stem are used in traditional Cameroonian traditional been m investigated. F. umbellata stem barks are usedbarks in Cameroonian medicine system medicine system for the treatment of conditions, many gynecological conditions, such as amenorrhea, for the treatment of many gynecological such as amenorrhea, dysmenorrhea as well as for dysmenorrhea as well as for addition, it is reported thatused figs for have menopausal complaints [16].menopausal In addition,complaints it is reported[16]. thatIn figs have been conventionally their been conventionally used for their therapeutic benefits as anticancer remedies [17,18]. Previous therapeutic benefits as anticancer remedies [17,18]. Previous studies showed that F. umbellata extracts, studies F. umbellata extracts, which contain 7-methylcoumarin (Table exhibited whichshowed containthat 7-methylcoumarin (Table 1) exhibited weak estrogenic effects in1) vitro and inweak vivo as estrogenic in constituent vitro and in asmost the of main bioactive constituent [19].ofSince most have of the maineffects bioactive [19].vivo Since ethnopharmacological usages F. umbellata ethnopharmacological usages of F. umbellata have been recommended in the literature to be taken been recommended in the literature to be taken into consideration when selecting plants for anticancer into consideration when selecting plants for anticancer theantitumor present work was of screenings [12], the present work was designed to assess screenings in vitro and[12], in vivo potentials designed to assess in vitro and cancer in vivo cells antitumor potentialstoofF.this plant. In vitro, various cancer cells this plant. In vitro, various were exposed umbellata aqueous (AE) and methanolic were exposed to F. umbellata aqueous (AE) and methanolic (MeOH) extracts, and the cytotoxicity, (MeOH) extracts, and the cytotoxicity, mechanism of cell death, cell cycle analysis, anti-migration and mechanism of cell death,reactive cell cycle analysis, anti-migration anti-invasion potential, reactive anti-invasion potential, oxygen species (ROS) levels, and mitochondrial transmembrane potential, oxygen species (ROS) levels, mitochondrial transmembrane potential, caspase activities and Bcl-2 caspase activities and Bcl-2 family proteins content were studied. In vivo, the chemopreventive effects family proteins content were studied. In vivo, the chemopreventive effects of F. umbellata extracts of F. umbellata extracts were investigated against 7,12 dimethylbenz(a)anthracene (DMBA)-induced were investigated against carcinogenesis 7,12 dimethylbenz(a)anthracene (DMBA)-induced mammary and ovarian mammary and ovarian in female Wistar rats. carcinogenesis in female Wistar rats. Table 1. General information on 7-methoxycoumarin isolated from F. umbellata. Table 1. General information on 7-methoxycoumarin isolated from F. umbellata. Chemical Names Chemical Names
CrystalColor Color Crystal
7-Methoxycoumarin 7-Methoxycoumarin Methylumbelliferone Methylumbelliferone 7-Methoxy-2H-chromen-2-one 7-Methoxy-2H-chromen-2-one
White White
Structure,Molecular Molecular Weight Weight and Structure, andFormula Formula
Molecular 176.1MM Molecular weight weight ==176.1 Molecular formula HH 1010 8O 33 Molecular formula==CC 8O
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2. Results 2. Results 2.1. Preliminary Phytochemical Analysis 2.1. Preliminary Phytochemical Analysis The phytochemical screening of F. umbellata AE and MeOH extracts showed that phenols, flavonoids, alkaloids, steroids, saponins, glycosides tannin were present in that both phenols, extracts. The phytochemical screening of F.cardiac umbellata AE andand MeOH extracts showed Their concentrations total phenols, flavonoids, flavonolsand andtannin alkaloids depicted in Table 2. flavonoids, alkaloids, in steroids, saponins, cardiac glycosides wereare present in both extracts. As observed in the table flavonoids were the mostflavonols abundantand secondary metabolites present in the Their concentrations in total phenols, flavonoids, alkaloids are depicted in Table 2. dried AE and MeOH extracts. Flavonols were found in both extracts, while alkaloids were the less As observed in the table flavonoids were the most abundant secondary metabolites present in the abundant. However, umbellata MeOH extract was found to contain more flavonoids, dried AE and MeOHF.extracts. Flavonols were found in both extracts, while alkaloids flavonols were the and less alkaloids than AE extract. abundant. However, F. umbellata MeOH extract was found to contain more flavonoids, flavonols and alkaloids than AE extract. Table 2. Quantitative analyses of selected phytochemicals present in F. umbellata extracts. Table 2. Quantitative analyses of selected phytochemicals present in F. umbellata extracts. Concentration of F. umbellata Extract N° Phytochemical Class Aqueous of F. umbellata Methanolic Concentration Extract ◦ Phytochemical Class 1 N Total phenols 540.21 ± 4.42 651.31 ± 12.03 Aqueous Methanolic 2 Flavonoids 268.25 ± 35.55 545.33 ± 99.39 1 Total phenols 540.21 ± 4.42 651.31 ± 12.03 3 Flavonols 78.22 ± 6.11 180.15 ± 10.74 2 Flavonoids 268.25 ± 35.55 545.33 ± 99.39 4 3 Alkaloids 60.21 14.38 120.21 6.04 Flavonols 78.22± ± 6.11 180.15 ± ±10.74 4 Alkaloids 60.21 ±in14.38 120.21 ± 6.04 Total phenols, flavonoids and flavonols are expressed mg eq quercetin/g of dried extract while Total phenols, flavonoids and flavonols areeq expressed in mgofeqdried quercetin/g of Data dried extract while alkaloids content alkaloids content is expressed in mg berberin/g extract. are represented as mean ± is expressed in mg eq berberin/g of dried extract. Data are represented as mean ± standard error of mean (SEM) of standard error of mean (SEM) of triplicates from at least three independent experiments. triplicates from at least three independent experiments.
2.2. Cytotoxicity of F. umbellata Extracts 2.2. Cytotoxicity of F. umbellata Extracts Figure 1 depicts the cytotoxicity response-curve of F. umbellata AE and MeOH extracts on breast Figure 1 depicts the cytotoxicity response-curve of F. umbellata AE and MeOH extracts on breast cancer cells (MCF-7 and MDA-MB-231) and one non-tumoral cell line (NIH-3T3). As shown in the cancer cells (MCF-7 and MDA-MB-231) and one non-tumoral cell line (NIH-3T3). As shown in the Figure, AE did not induce significant cytotoxicity up to 300 μg/mL, while the MeOH extract showed Figure, AE did not induce significant cytotoxicity up to 300 µg/mL, while the MeOH extract showed concentration-dependent cytotoxicity after 24 h of incubation and this effect was more pronounced concentration-dependent cytotoxicity after 24 h of incubation and this effect was more pronounced in in estrogen receptor-negative cells (MDA-MB-231, CC50 = 180 μg/mL) (Table 3A). Further, the 3 estrogen receptor-negative cells (MDA-MB-231, CC50 = 180 µg/mL) (Table 3A). Further, the 3 fractions fractions from F. umbellata MeOH extract as well as 7-methoxycoumarin were assessed for their from F. umbellata MeOH extract as well as 7-methoxycoumarin were assessed for their cytotoxicity in cytotoxicity in the same cell lines. Residue fraction (FU-R) and 7-methoxycoumarin (MC) did not the same cell lines. Residue fraction (FU-R) and 7-methoxycoumarin (MC) did not showed cytotoxicity showed cytotoxicity up to 300 μg/mL or 300 μM, respectively, in the tested cell lines, whereas up to 300 µg/mL or 300 µM, respectively, in the tested cell lines, whereas dichloromethane fraction dichloromethane fraction (FU-DCM) exhibited cytotoxicity at concentrations close to those of MeOH (FU-DCM) exhibited cytotoxicity at concentrations close to those of MeOH extract (Table 3A). To follow extract (Table 3A). To follow up, the cytotoxicity of F. umbellata MeOH extract and DCM fraction up, the cytotoxicity of F. umbellata MeOH extract and DCM fraction (FU-DCM) were assessed in five (FU-DCM) were assessed in five other cell lines (SF-295, 4T1, HUVEC, MCR-5, SK-MEL-28 and HCC other cell lines (SF-295, 4T1, HUVEC, MCR-5, SK-MEL-28 and HCC 1995). Their CC50 were found to 1995). Their CC50 were found to be quasi-similar in these cell lines (Table 3B), affording a selectivity be quasi-similar in these cell lines (Table 3B), affording a selectivity index around two in human and index around two in human and one in murine cell lines (Table 3C). Since F. umbellata MeOH extract one in murine cell lines (Table 3C). Since F. umbellata MeOH extract and FU-DCM fraction have the and FU-DCM fraction have the same cytotoxic activities, the understanding of underlying same cytotoxic activities, the understanding of underlying mechanisms of this plant was focused on mechanisms of this plant was focused on MeOH extract. MeOH extract.
Figure 1. Concentration-dependent cytotoxicity of F. umbellata aqueous (AE) and methanolic extracts (MeOH) in breast breast cancer cancerand andnon-tumoral non-tumoralcell celllines. lines.MCF-7 MCF-7 (A); MDA-MB-231 NIH-3T3 (A); MDA-MB-231 (B);(B); andand NIH-3T3 (C) (C) cells were incubated with increasing concentrations (50–300 µg/mL) of extracts for 24 h and cell cells were incubated with increasing concentrations (50–300 μg/mL) of extracts for 24 h and viability was evaluated by Alamar Alamarblue blue(resazurin) (resazurin)assay. assay.The The results expressed in percentage as evaluated by results areare expressed in percentage as the the mean ± SEM. mean ± SEM.
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Table 3. CC50 values of F. umbellata extracts in tumoral and non-tumoral cell lines. CC50 (µg/mL or µM) MeOH MCF-7 250 A MDA-MB-231 180 NIH-3T3 297
AE >300 >300 >300
FU-Hex >300 190 270
FU-DCM 197 180 215
FU-R >300 >300 >300
MC >300 >300 >300
CC50 (µg/mL) B
MeOH FU-DCM
SF-295 237 252
HUVEC 449 440
4T1 283 245
MRC-5 378 370
SK-MEL-28 HCC 1954 185 192 176 162
Selectivity Index C
MeOH FU-DCM
HUVEC/MCF-7 1.6 2.23
MRC-5/MDA-MB-231 2.08 2.05
NIH-3T3/4T1 1.05 0.88
CC50 , Concentration of F. umbellata extracts which results in 50% of cell viability; Selectivity Index is equal to CC50 of F. umbellata extracts on non-tumoral cell lines (NIH/3T3, MCR-5 and HUVEC) divided by CC50 determined for cancer cells (MCF-7, MDA-MB-231 and 4T1). (A) MCF-7, MDA-MB-231 and NIH-3T3 cells were exposed to F. umbellata methanol (MeOH) and aqueous extracts (AE); hexane (FU-Hex), dichloromethane (FU-DCM) and residue (FU-R) fractions from F. umbellata MeOH extract as well as 7-methoxycoumarin (MC) at two concentrations (100 and 300 µg/mL or 100 and 300 µM) for the screening; (B) CC50 of MeOH extract and FU-DCM fraction in SF-295, 4T1, HUVEC, MCR-5, SK-MEL-28 and HCC 1954 cell lines; C, selectivity index of MeOH and FU-DCM in human and murine cell lines.
2.3. Effects on Cell Death Mechanism Apoptosis was evaluated by the qualitative acridine orange (AO)/ethidium bromide (EB) staining in this study. AO is absorbed by cells with intact or damaged membranes, and emits green fluorescence when it intercalates DNA. Ethidium bromide is taken only by cells with damaged membranes and emits red fluorescence fixed on DNA. Figure 2A and Figure S1A permit to distinguish three types of cells according to the fluorescence emission and the morphological aspect of chromatin condensation in the stained nuclei. Intact membrane cells (IMC) have uniform bright green nuclei with an organized structure. Apoptotic cells (Ap), which have green nuclei with condensed or fragmented chromatin and necrotic cells (Ne) have uniformly orange to red nuclei with an organized structure. It has been Int. J. Mol. Sci. 2017, 18, 1073 5 of 27in observed a chromatin condensation characteristic to apoptosis following F. umbelleta MeOH extract MDA-MB-231 (Figure 2A) and MCF-7 (Figure S1A) cells lines.
Figure 2. Effect of F. umbellata extracts on cancer cell death. Representative fluorescence microscopic Figure 2. Effect of F. umbellata extracts on cancer cell death. Representative fluorescence microscopic images (400×) of cells double-stained with acridine orange (0.3 mg/mL) and ethidium bromide images (400×) of cells double-stained with acridine orange (0.3 mg/mL) and ethidium bromide (1 mg/mL) (A); and dot plot representative of one experiment of apoptosis measurement by (1 mg/mL) (A); and dot plot representative of one experiment of apoptosis measurement by Annexin-V-FITC/PI staining (B). MDA-MB-231 cells were treated for 24 h to F. umbellata extracts at Annexin-V-FITC/PI staining (B). MDA-MB-231 cells were treated for 24 h to F. umbellata extracts concentrations of 45, 90 and 180 μg/mL. (C) A graph showing the mean ± SEM of three independent at concentrations of 45, 90 and 180 µg/mL. (C) A graph showing the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared with control. IMC, intact membrane experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared with control. IMC, intact membrane cell; Ap, apoptotic cells; Ne, necrotic cells. cell; Ap, apoptotic cells; Ne, necrotic cells.
2.4. Effects on Cell Cycle Flow cytometry was used to determine the effects of F. umbellata MeOH extract on the cell cycle progression of MCF-7 and MDA-MB-231 cells. It did not induce significant change in both MDA-MB-231 (Figure 3) and MCF-7 (Figure S2) cell cycle after 12, 24 and 48 h of treatment. Since effects of F. umbellata MeOH extract were the same in MDA-MB-231 and MCF-7 cells, we used
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Furthermore, the cell death mechanisms induced by F. umbellata MeOH extract were evaluated throughFigure flow cytometry. depicted in dot plotscell (Figure and Figurefluorescence S1B), the viable cells had low 2. Effect of F.As umbellata extracts on cancer death.2B Representative microscopic images (400×) cellsPIdouble-stained acridine orange mg/mL) andFITC ethidium bromide but low FITC fluorescence andoflow fluorescence;with early apoptotic cell(0.3 showed high fluorescence, (1 mg/mL)The (A); percentages and dot plot of representative of one experiment apoptosis by PI fluorescence. apoptotic and necrotic cellsofwere 1.8% measurement and 1.7%, respectively, Annexin-V-FITC/PI staining (B). MDA-MB-231increase cells wereintreated for 24 h to F.of umbellata extracts at with the in control cells. The concentration-dependent the percentage apoptotic cells concentrations of 45, 90 and 180 μg/mL. (C) A graph showing the mean ± SEM of three independent maximum of 33.9% at 180 µg/mL of F. umbellata MeOH extract suggests that it induced apoptosis in experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared with control. IMC, intact membrane the MDA-MB-231 (Figure 2C) and MCF-7 (Figure S1C) cells. cell; Ap, apoptotic cells; Ne, necrotic cells.
2.4. Effects on Cell Cycle
2.4. Effects on Cell Cycle
Flow cytometry was the effects effectsofofF.F.umbellata umbellata MeOH extract on cell the cycle cell cycle Flow cytometry wasused usedtotodetermine determine the MeOH extract on the progression of MCF-7 and MDA-MB-231 cells. It did not induce significant change in both MDA-MB-231 progression of MCF-7 and MDA-MB-231 cells. It did not induce significant change in both (Figure 3) and MCF-7 (Figure S2)MCF-7 cell cycle afterS2) 12,cell 24 and h of12, treatment. of F.Since umbellata MDA-MB-231 (Figure 3) and (Figure cycle48after 24 and 48Since h of effects treatment. MeOH extract the MeOH same in MDA-MB-231 and MCF-7 cells, weand used MDA-MB-231 cells for effects of F. were umbellata extract were the same in MDA-MB-231 MCF-7 cells, we used MDA-MB-231 cells for evaluating anticancer effects of extract. evaluating anticancer effects of extract.
Figure 3. Effect umbellataextracts extractson on cell cell cycle cycle distribution cells afterafter different Figure 3. Effect of of F. F. umbellata distributionininMDA-MB-231 MDA-MB-231 cells different times of incubation:12 12 hh (A); 24 4848 h (C). Cells werewere treated for 24for h with 90 and times of incubation: 24hh(B); (B);and and h (C). Cells treated 24 h45,with 45, 180 90 and with PI.PI. Following flow cytometry, cellular DNA profile was was 180 μg/mL µg/mLofofextracts extractsand andstaining staining with Following flow cytometry, cellular DNA profile analyzed using software WinMDI Data represent the percentageofofcell cellcounts countsinineach eachcell cellcycle analyzed using thethe software WinMDI 2.9.2.9. Data represent the percentage cycle phase. The results are expressed as the mean ± SEM of three independent experiments. No phase. The results are expressed as the mean ± SEM of three independent experiments. No significant significant change was noted. change was noted.
2.5. Effects on Cell Migration and Invasion
2.5. Effects on Cell Migration and Invasion
It was observed a significant and concentration-dependent inhibition of MDA-MB-231 cells It was observed a significant concentration-dependent MDA-MB-231 migration with F. umbellata MeOH and extract at concentration of 90 and inhibition 180 μg/mL of after 24 and 48 h ofcells migration with F. umbellata MeOH at determine concentration of 90 of and µg/mL afterextract 24 and exposition (Figure 4). Further, we extract sought to the ability F. 180 umbellata MeOH to 48 h inhibit MDA-MB-231 invasion. It was to observed a significant (p
Molecular Sciences Article
Ficus umbellata Vahl. (Moraceae) Stem Bark Extracts Exert Antitumor Activities In Vitro and In Vivo Kevine Kamga Silihe 1 , Stéphane Zingue 2,3,4,5 , Evelyn Winter 4 , Charline Florence Awounfack 3 , Anupam Bishayee 6, *, Nishil N. Desai 6 , Leônidas João Mello Jr. 4 , Thomas Michel 7 , Francine Nzufo Tankeu 1 , Derek Tantoh Ndinteh 5 , Sara Honorine Riwom 1 , Dieudonné Njamen 3,5 and Tânia Beatriz Creczynski-Pasa 4, * 1 2 3 4 5 6 7
*
Department of Biochemistry, Faculty of Sciences, University of Yaounde 1, Yaounde 812, Cameroon; [email protected] (K.K.S.); [email protected] (F.N.T.); [email protected] (S.H.R.) Department of Life and Earth Sciences, Higher Teachers’ Training College, University of Maroua, Maroua 55, Cameroon; [email protected] Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé 1, Yaounde 812, Cameroon; [email protected] (C.F.A.); [email protected] (D.N.) Department of Pharmaceutical Sciences and Department of Biochemistry, Federal University of Santa Catarina, Florianópolis 88040-900, Brazil; [email protected] (E.W.); [email protected] (L.J.M.) Department of Applied Chemistry, Faculty of Sciences, University of Johannesburg, Doornfontein 2028, South Africa; [email protected] Department of Pharmaceutical Sciences, College of Pharmacy, Larkin University, Miami, FL 33169, USA; [email protected] Institute of Chemistry of Nice, Faculty of Sciences, University Côte d’Azur, Nice F-06108 Nice CEDEX 2, France; [email protected] Correspondence: [email protected] or [email protected] (A.B.); [email protected] (T.B.C.-P.); Tel.: +1-305-760-7511 (A.B.)
Academic Editor: Leticia M. Estevinho Received: 29 March 2017; Accepted: 3 May 2017; Published: 23 May 2017
Abstract: A Ficus umbellata is used to treat cancer. The present work was therefore designed to assess antitumor potentials of F. umbellata extracts in nine different cell lines. Cell cycle, apoptosis, cell migration/invasion, levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), caspases activities as well as Bcl-2 and Bcl-xL protein content were assessed in MDA-MB-231 cells. The 7,12-dimethylbenz(a)anthracene (DMBA)-induced carcinogenesis in rats were also used to investigate antitumor potential of F. umbellata extracts. The F. umbellata methanol extract exhibited a CC50 of 180 µg/mL in MDA-MB-231 cells after 24 h. It induced apoptosis in MCF-7 and MDAMB-231 cells, while it did not alter their cell cycle phases. Further, it induced a decrease in MMP, an increase in ROS levels and caspases activities as well as a downregulation in Bcl-2 and Bcl-xL protein contents in MDA-MB-231 cells. In vivo, F. umbellata aqueous (200 mg/kg) and methanol (50 mg/kg) extracts significantly (p < 0.001) reduced ovarian tumor incidence (10%), total tumor burden (58% and 46%, respectively), average tumor weight (57.8% and 45.6%, respectively) as compared to DMBA control group. These results suggest antitumor potential of F. umbellata constituents possibly due to apoptosis induction mediated through ROS-dependent mitochondrial pathway. Keywords: Ficus umbellata; apoptosis; estrogen-dependent tumors; DMBA; cell invasion
1. Introduction Cancer is a group of diseases characterized by the unregulated proliferation of abnormal cells that invade and disrupt surrounding tissues [1]. It is a source of significant morbidity worldwide, responsible for over 8.2 million deaths per year and a serious public health problem in both developed Int. J. Mol. Sci. 2017, 18, 1073; doi:10.3390/ijms18061073
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 1073 Int. J. Mol. Sci. 2017, 18, 1073
2 of 27 2 of 27
responsible for over 8.2 million deaths per year and a serious public health problem in both developed and developing countries [2]. and Breast and gynecological cancers (e.g., ovarian and uterus) and developing countries [2]. Breast gynecological cancers (e.g., ovarian and uterus) are the aremajor the major cause of cancer death in women of developing countries, and represent 19% cancers cause of cancer death in women of developing countries, and represent 19%ofof cancers worldwide World Health Organization, the incidence of breast and worldwide[3]. [3]. According According totothethe World Health Organization, the incidence of breast and gynecological gynecological cancers is predicted to increase in the future and 70% new cases will be observed in cancers is predicted to increase in the future and 70% new cases will be observed in developing developing countries [2]. In Africa, the most common cancers in women are breast and cervical ones countries [2]. In Africa, the most common cancers in women are breast and cervical ones [4]. However, [4].endometrial However, endometrial ovarian cancers are also routinely incidence of ovarian and ovarian and cancers are also routinely observed. Theobserved. incidenceThe of ovarian cancer increases cancer increases worldwide by about 10-fold in women during the perito post-menopausal period, worldwide by about 10-fold in women during the peri- to post-menopausal period, when compared when compared to premenopausal women With per 3500year, deaths year, cancer the ovarian is the of to premenopausal women [5]. With 3500[5]. deaths the per ovarian is thecancer most lethal most lethal of gynecologic malignancies and a relatively uncommon cancer, making testing of drugs gynecologic malignancies and a relatively uncommon cancer, making testing of drugs for primary forprevention primary prevention difficult [6,7]. difficult [6,7]. Nowadays, regimensagainst against breast and gynecological cancersradiotherapy, include Nowadays,therapeutic therapeutic regimens breast and gynecological cancers include radiotherapy, chemotherapy, immunotherapy and hormonal therapies, and, in advanced stages of chemotherapy, immunotherapy and hormonal therapies, and, in advanced stages of the disease, surgery theintervention disease, surgery intervention can be required [8,9]. Despite substantial improvements in can be required [8,9]. Despite substantial improvements in survival, the management survival, theis management is limited to its high scarcity of diagnosis of cancer limited due toofitscancer high cost, scarcity due of diagnosis and cost, treatment, limited access to and health treatment, limited access to health services found in African countries [10], and most importantly the as services found in African countries [10], and most importantly the problem of resistance as well problem of resistance as well as high systemic toxicity ofdrugs available drugs [11]. The is high systemic toxicity of available chemotherapeutic [11].chemotherapeutic The need for primary prevention need for primary prevention is worthwhile and novel efficacious, non-toxic and cost-effective worthwhile and novel efficacious, non-toxic and cost-effective therapeutic agents should be developed. therapeutic should be and developed. Moreover, epidemiological and laboratory studies have Moreover,agents epidemiological laboratory studies have provided evidence that naturally occurring provided that naturally occurring dietary or plantagainst components may exert[12–14]. protective effects dietary evidence or plant components may exert protective effects various cancers Increasingly, against various cancers [12–14]. Increasingly, more medicinal plants are being screened for more medicinal plants are being screened for anticancer properties and may provide effective anticancer properties and may provide effective therapeutic agents. therapeutic agents. The primary prevention of of breast and gynecological cancers remains an an important issue forfor The primary prevention breast and gynecological cancers remains important issue developing countries, where 80% of the population still resorts to traditional medicine for their developing countries, where 80% of the population still resorts to traditional medicine for their primary primary care In thisFicus scope, Ficus umbellata, a plant in subtropical regions,up growing health health care [15]. In[15]. this scope, umbellata, a plant found in found subtropical regions, growing to 6–10 m uphas to 6–10 has been investigated. F. umbellata stem are used in traditional Cameroonian traditional been m investigated. F. umbellata stem barks are usedbarks in Cameroonian medicine system medicine system for the treatment of conditions, many gynecological conditions, such as amenorrhea, for the treatment of many gynecological such as amenorrhea, dysmenorrhea as well as for dysmenorrhea as well as for addition, it is reported thatused figs for have menopausal complaints [16].menopausal In addition,complaints it is reported[16]. thatIn figs have been conventionally their been conventionally used for their therapeutic benefits as anticancer remedies [17,18]. Previous therapeutic benefits as anticancer remedies [17,18]. Previous studies showed that F. umbellata extracts, studies F. umbellata extracts, which contain 7-methylcoumarin (Table exhibited whichshowed containthat 7-methylcoumarin (Table 1) exhibited weak estrogenic effects in1) vitro and inweak vivo as estrogenic in constituent vitro and in asmost the of main bioactive constituent [19].ofSince most have of the maineffects bioactive [19].vivo Since ethnopharmacological usages F. umbellata ethnopharmacological usages of F. umbellata have been recommended in the literature to be taken been recommended in the literature to be taken into consideration when selecting plants for anticancer into consideration when selecting plants for anticancer theantitumor present work was of screenings [12], the present work was designed to assess screenings in vitro and[12], in vivo potentials designed to assess in vitro and cancer in vivo cells antitumor potentialstoofF.this plant. In vitro, various cancer cells this plant. In vitro, various were exposed umbellata aqueous (AE) and methanolic were exposed to F. umbellata aqueous (AE) and methanolic (MeOH) extracts, and the cytotoxicity, (MeOH) extracts, and the cytotoxicity, mechanism of cell death, cell cycle analysis, anti-migration and mechanism of cell death,reactive cell cycle analysis, anti-migration anti-invasion potential, reactive anti-invasion potential, oxygen species (ROS) levels, and mitochondrial transmembrane potential, oxygen species (ROS) levels, mitochondrial transmembrane potential, caspase activities and Bcl-2 caspase activities and Bcl-2 family proteins content were studied. In vivo, the chemopreventive effects family proteins content were studied. In vivo, the chemopreventive effects of F. umbellata extracts of F. umbellata extracts were investigated against 7,12 dimethylbenz(a)anthracene (DMBA)-induced were investigated against carcinogenesis 7,12 dimethylbenz(a)anthracene (DMBA)-induced mammary and ovarian mammary and ovarian in female Wistar rats. carcinogenesis in female Wistar rats. Table 1. General information on 7-methoxycoumarin isolated from F. umbellata. Table 1. General information on 7-methoxycoumarin isolated from F. umbellata. Chemical Names Chemical Names
CrystalColor Color Crystal
7-Methoxycoumarin 7-Methoxycoumarin Methylumbelliferone Methylumbelliferone 7-Methoxy-2H-chromen-2-one 7-Methoxy-2H-chromen-2-one
White White
Structure,Molecular Molecular Weight Weight and Structure, andFormula Formula
Molecular 176.1MM Molecular weight weight ==176.1 Molecular formula HH 1010 8O 33 Molecular formula==CC 8O
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2. Results 2. Results 2.1. Preliminary Phytochemical Analysis 2.1. Preliminary Phytochemical Analysis The phytochemical screening of F. umbellata AE and MeOH extracts showed that phenols, flavonoids, alkaloids, steroids, saponins, glycosides tannin were present in that both phenols, extracts. The phytochemical screening of F.cardiac umbellata AE andand MeOH extracts showed Their concentrations total phenols, flavonoids, flavonolsand andtannin alkaloids depicted in Table 2. flavonoids, alkaloids, in steroids, saponins, cardiac glycosides wereare present in both extracts. As observed in the table flavonoids were the mostflavonols abundantand secondary metabolites present in the Their concentrations in total phenols, flavonoids, alkaloids are depicted in Table 2. dried AE and MeOH extracts. Flavonols were found in both extracts, while alkaloids were the less As observed in the table flavonoids were the most abundant secondary metabolites present in the abundant. However, umbellata MeOH extract was found to contain more flavonoids, dried AE and MeOHF.extracts. Flavonols were found in both extracts, while alkaloids flavonols were the and less alkaloids than AE extract. abundant. However, F. umbellata MeOH extract was found to contain more flavonoids, flavonols and alkaloids than AE extract. Table 2. Quantitative analyses of selected phytochemicals present in F. umbellata extracts. Table 2. Quantitative analyses of selected phytochemicals present in F. umbellata extracts. Concentration of F. umbellata Extract N° Phytochemical Class Aqueous of F. umbellata Methanolic Concentration Extract ◦ Phytochemical Class 1 N Total phenols 540.21 ± 4.42 651.31 ± 12.03 Aqueous Methanolic 2 Flavonoids 268.25 ± 35.55 545.33 ± 99.39 1 Total phenols 540.21 ± 4.42 651.31 ± 12.03 3 Flavonols 78.22 ± 6.11 180.15 ± 10.74 2 Flavonoids 268.25 ± 35.55 545.33 ± 99.39 4 3 Alkaloids 60.21 14.38 120.21 6.04 Flavonols 78.22± ± 6.11 180.15 ± ±10.74 4 Alkaloids 60.21 ±in14.38 120.21 ± 6.04 Total phenols, flavonoids and flavonols are expressed mg eq quercetin/g of dried extract while Total phenols, flavonoids and flavonols areeq expressed in mgofeqdried quercetin/g of Data dried extract while alkaloids content alkaloids content is expressed in mg berberin/g extract. are represented as mean ± is expressed in mg eq berberin/g of dried extract. Data are represented as mean ± standard error of mean (SEM) of standard error of mean (SEM) of triplicates from at least three independent experiments. triplicates from at least three independent experiments.
2.2. Cytotoxicity of F. umbellata Extracts 2.2. Cytotoxicity of F. umbellata Extracts Figure 1 depicts the cytotoxicity response-curve of F. umbellata AE and MeOH extracts on breast Figure 1 depicts the cytotoxicity response-curve of F. umbellata AE and MeOH extracts on breast cancer cells (MCF-7 and MDA-MB-231) and one non-tumoral cell line (NIH-3T3). As shown in the cancer cells (MCF-7 and MDA-MB-231) and one non-tumoral cell line (NIH-3T3). As shown in the Figure, AE did not induce significant cytotoxicity up to 300 μg/mL, while the MeOH extract showed Figure, AE did not induce significant cytotoxicity up to 300 µg/mL, while the MeOH extract showed concentration-dependent cytotoxicity after 24 h of incubation and this effect was more pronounced concentration-dependent cytotoxicity after 24 h of incubation and this effect was more pronounced in in estrogen receptor-negative cells (MDA-MB-231, CC50 = 180 μg/mL) (Table 3A). Further, the 3 estrogen receptor-negative cells (MDA-MB-231, CC50 = 180 µg/mL) (Table 3A). Further, the 3 fractions fractions from F. umbellata MeOH extract as well as 7-methoxycoumarin were assessed for their from F. umbellata MeOH extract as well as 7-methoxycoumarin were assessed for their cytotoxicity in cytotoxicity in the same cell lines. Residue fraction (FU-R) and 7-methoxycoumarin (MC) did not the same cell lines. Residue fraction (FU-R) and 7-methoxycoumarin (MC) did not showed cytotoxicity showed cytotoxicity up to 300 μg/mL or 300 μM, respectively, in the tested cell lines, whereas up to 300 µg/mL or 300 µM, respectively, in the tested cell lines, whereas dichloromethane fraction dichloromethane fraction (FU-DCM) exhibited cytotoxicity at concentrations close to those of MeOH (FU-DCM) exhibited cytotoxicity at concentrations close to those of MeOH extract (Table 3A). To follow extract (Table 3A). To follow up, the cytotoxicity of F. umbellata MeOH extract and DCM fraction up, the cytotoxicity of F. umbellata MeOH extract and DCM fraction (FU-DCM) were assessed in five (FU-DCM) were assessed in five other cell lines (SF-295, 4T1, HUVEC, MCR-5, SK-MEL-28 and HCC other cell lines (SF-295, 4T1, HUVEC, MCR-5, SK-MEL-28 and HCC 1995). Their CC50 were found to 1995). Their CC50 were found to be quasi-similar in these cell lines (Table 3B), affording a selectivity be quasi-similar in these cell lines (Table 3B), affording a selectivity index around two in human and index around two in human and one in murine cell lines (Table 3C). Since F. umbellata MeOH extract one in murine cell lines (Table 3C). Since F. umbellata MeOH extract and FU-DCM fraction have the and FU-DCM fraction have the same cytotoxic activities, the understanding of underlying same cytotoxic activities, the understanding of underlying mechanisms of this plant was focused on mechanisms of this plant was focused on MeOH extract. MeOH extract.
Figure 1. Concentration-dependent cytotoxicity of F. umbellata aqueous (AE) and methanolic extracts (MeOH) in breast breast cancer cancerand andnon-tumoral non-tumoralcell celllines. lines.MCF-7 MCF-7 (A); MDA-MB-231 NIH-3T3 (A); MDA-MB-231 (B);(B); andand NIH-3T3 (C) (C) cells were incubated with increasing concentrations (50–300 µg/mL) of extracts for 24 h and cell cells were incubated with increasing concentrations (50–300 μg/mL) of extracts for 24 h and viability was evaluated by Alamar Alamarblue blue(resazurin) (resazurin)assay. assay.The The results expressed in percentage as evaluated by results areare expressed in percentage as the the mean ± SEM. mean ± SEM.
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Table 3. CC50 values of F. umbellata extracts in tumoral and non-tumoral cell lines. CC50 (µg/mL or µM) MeOH MCF-7 250 A MDA-MB-231 180 NIH-3T3 297
AE >300 >300 >300
FU-Hex >300 190 270
FU-DCM 197 180 215
FU-R >300 >300 >300
MC >300 >300 >300
CC50 (µg/mL) B
MeOH FU-DCM
SF-295 237 252
HUVEC 449 440
4T1 283 245
MRC-5 378 370
SK-MEL-28 HCC 1954 185 192 176 162
Selectivity Index C
MeOH FU-DCM
HUVEC/MCF-7 1.6 2.23
MRC-5/MDA-MB-231 2.08 2.05
NIH-3T3/4T1 1.05 0.88
CC50 , Concentration of F. umbellata extracts which results in 50% of cell viability; Selectivity Index is equal to CC50 of F. umbellata extracts on non-tumoral cell lines (NIH/3T3, MCR-5 and HUVEC) divided by CC50 determined for cancer cells (MCF-7, MDA-MB-231 and 4T1). (A) MCF-7, MDA-MB-231 and NIH-3T3 cells were exposed to F. umbellata methanol (MeOH) and aqueous extracts (AE); hexane (FU-Hex), dichloromethane (FU-DCM) and residue (FU-R) fractions from F. umbellata MeOH extract as well as 7-methoxycoumarin (MC) at two concentrations (100 and 300 µg/mL or 100 and 300 µM) for the screening; (B) CC50 of MeOH extract and FU-DCM fraction in SF-295, 4T1, HUVEC, MCR-5, SK-MEL-28 and HCC 1954 cell lines; C, selectivity index of MeOH and FU-DCM in human and murine cell lines.
2.3. Effects on Cell Death Mechanism Apoptosis was evaluated by the qualitative acridine orange (AO)/ethidium bromide (EB) staining in this study. AO is absorbed by cells with intact or damaged membranes, and emits green fluorescence when it intercalates DNA. Ethidium bromide is taken only by cells with damaged membranes and emits red fluorescence fixed on DNA. Figure 2A and Figure S1A permit to distinguish three types of cells according to the fluorescence emission and the morphological aspect of chromatin condensation in the stained nuclei. Intact membrane cells (IMC) have uniform bright green nuclei with an organized structure. Apoptotic cells (Ap), which have green nuclei with condensed or fragmented chromatin and necrotic cells (Ne) have uniformly orange to red nuclei with an organized structure. It has been Int. J. Mol. Sci. 2017, 18, 1073 5 of 27in observed a chromatin condensation characteristic to apoptosis following F. umbelleta MeOH extract MDA-MB-231 (Figure 2A) and MCF-7 (Figure S1A) cells lines.
Figure 2. Effect of F. umbellata extracts on cancer cell death. Representative fluorescence microscopic Figure 2. Effect of F. umbellata extracts on cancer cell death. Representative fluorescence microscopic images (400×) of cells double-stained with acridine orange (0.3 mg/mL) and ethidium bromide images (400×) of cells double-stained with acridine orange (0.3 mg/mL) and ethidium bromide (1 mg/mL) (A); and dot plot representative of one experiment of apoptosis measurement by (1 mg/mL) (A); and dot plot representative of one experiment of apoptosis measurement by Annexin-V-FITC/PI staining (B). MDA-MB-231 cells were treated for 24 h to F. umbellata extracts at Annexin-V-FITC/PI staining (B). MDA-MB-231 cells were treated for 24 h to F. umbellata extracts concentrations of 45, 90 and 180 μg/mL. (C) A graph showing the mean ± SEM of three independent at concentrations of 45, 90 and 180 µg/mL. (C) A graph showing the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared with control. IMC, intact membrane experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared with control. IMC, intact membrane cell; Ap, apoptotic cells; Ne, necrotic cells. cell; Ap, apoptotic cells; Ne, necrotic cells.
2.4. Effects on Cell Cycle Flow cytometry was used to determine the effects of F. umbellata MeOH extract on the cell cycle progression of MCF-7 and MDA-MB-231 cells. It did not induce significant change in both MDA-MB-231 (Figure 3) and MCF-7 (Figure S2) cell cycle after 12, 24 and 48 h of treatment. Since effects of F. umbellata MeOH extract were the same in MDA-MB-231 and MCF-7 cells, we used
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Furthermore, the cell death mechanisms induced by F. umbellata MeOH extract were evaluated throughFigure flow cytometry. depicted in dot plotscell (Figure and Figurefluorescence S1B), the viable cells had low 2. Effect of F.As umbellata extracts on cancer death.2B Representative microscopic images (400×) cellsPIdouble-stained acridine orange mg/mL) andFITC ethidium bromide but low FITC fluorescence andoflow fluorescence;with early apoptotic cell(0.3 showed high fluorescence, (1 mg/mL)The (A); percentages and dot plot of representative of one experiment apoptosis by PI fluorescence. apoptotic and necrotic cellsofwere 1.8% measurement and 1.7%, respectively, Annexin-V-FITC/PI staining (B). MDA-MB-231increase cells wereintreated for 24 h to F.of umbellata extracts at with the in control cells. The concentration-dependent the percentage apoptotic cells concentrations of 45, 90 and 180 μg/mL. (C) A graph showing the mean ± SEM of three independent maximum of 33.9% at 180 µg/mL of F. umbellata MeOH extract suggests that it induced apoptosis in experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared with control. IMC, intact membrane the MDA-MB-231 (Figure 2C) and MCF-7 (Figure S1C) cells. cell; Ap, apoptotic cells; Ne, necrotic cells.
2.4. Effects on Cell Cycle
2.4. Effects on Cell Cycle
Flow cytometry was the effects effectsofofF.F.umbellata umbellata MeOH extract on cell the cycle cell cycle Flow cytometry wasused usedtotodetermine determine the MeOH extract on the progression of MCF-7 and MDA-MB-231 cells. It did not induce significant change in both MDA-MB-231 progression of MCF-7 and MDA-MB-231 cells. It did not induce significant change in both (Figure 3) and MCF-7 (Figure S2)MCF-7 cell cycle afterS2) 12,cell 24 and h of12, treatment. of F.Since umbellata MDA-MB-231 (Figure 3) and (Figure cycle48after 24 and 48Since h of effects treatment. MeOH extract the MeOH same in MDA-MB-231 and MCF-7 cells, weand used MDA-MB-231 cells for effects of F. were umbellata extract were the same in MDA-MB-231 MCF-7 cells, we used MDA-MB-231 cells for evaluating anticancer effects of extract. evaluating anticancer effects of extract.
Figure 3. Effect umbellataextracts extractson on cell cell cycle cycle distribution cells afterafter different Figure 3. Effect of of F. F. umbellata distributionininMDA-MB-231 MDA-MB-231 cells different times of incubation:12 12 hh (A); 24 4848 h (C). Cells werewere treated for 24for h with 90 and times of incubation: 24hh(B); (B);and and h (C). Cells treated 24 h45,with 45, 180 90 and with PI.PI. Following flow cytometry, cellular DNA profile was was 180 μg/mL µg/mLofofextracts extractsand andstaining staining with Following flow cytometry, cellular DNA profile analyzed using software WinMDI Data represent the percentageofofcell cellcounts countsinineach eachcell cellcycle analyzed using thethe software WinMDI 2.9.2.9. Data represent the percentage cycle phase. The results are expressed as the mean ± SEM of three independent experiments. No phase. The results are expressed as the mean ± SEM of three independent experiments. No significant significant change was noted. change was noted.
2.5. Effects on Cell Migration and Invasion
2.5. Effects on Cell Migration and Invasion
It was observed a significant and concentration-dependent inhibition of MDA-MB-231 cells It was observed a significant concentration-dependent MDA-MB-231 migration with F. umbellata MeOH and extract at concentration of 90 and inhibition 180 μg/mL of after 24 and 48 h ofcells migration with F. umbellata MeOH at determine concentration of 90 of and µg/mL afterextract 24 and exposition (Figure 4). Further, we extract sought to the ability F. 180 umbellata MeOH to 48 h inhibit MDA-MB-231 invasion. It was to observed a significant (p
