pharmaceuticals

Article Potent Cytotoxicity of Four Cameroonian Plant Extracts on Different Cancer Cell Lines

Ahmed Somaida 1 , Imran Tariq 1,2 , Ghazala Ambreen 1 , Ahmed Mohamed Abdelsalam 1,3, Abdallah Mohamed Ayoub 1,4, Matthias Wojcik 1, Jean Paul Dzoyem 5,* and Udo Bakowsky 1,*

1 Department of Pharmaceutics and Biopharmaceutics, University of Marburg, 35037 Marburg, Germany; somaida@staff.uni-marburg.de (A.S.); [email protected] (I.T.); [email protected] (G.A.); [email protected] (A.M.A.); ayoub@staff.uni-marburg.de (A.M.A.); [email protected] (M.W.) 2 Punjab University College of Pharmacy, University of the Punjab, Lahore 54000, Pakistan 3 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Al-Azhar University, Assiut 11865, Egypt 4 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt 5 Department of Biochemistry, Faculty of Science, University of Dschang. P.O Box. 67 Dschang, Cameroon * Correspondence: [email protected] (J.P.D.); [email protected] (U.B.)



Received: 5 September 2020; Accepted: 29 October 2020; Published: 31 October 2020


Abstract: In this study, the potential cytotoxicity of four plant extracts originated from Cameroon: Xylopia aethiopica (XA), Imperata cylindrica (IC), Echinops giganteus (EG) and Dorstenia psilurus (DP) were examined in vitro. We tested the anti-proliferative activity of the methanolic extracts of these compounds using MTT assay on seven different human cancer cell lines: HeLa, MDA-MB-231, A549, HepG2, U-87, SK-OV-3 and HL60. Induction of cell death was assessed by cell cycle analysis, apoptosis was determined by Annexin V-FITC binding and caspase 3/7 activity. As well, changes in mitochondrial membrane potential (MMP) and cell migration were tested. The genetic toxicity, using the alkaline comet assay, was evaluated. The studied extracts inhibited the cell proliferation of all tested cancer cell lines with concentration dependent effect over time. All of these extracts mainly induced apoptosis of HeLa cells by the accumulation of hypodiploid cells in the sub-G0/G1 phase and increasing the activity of caspase 3/7, as well they showed potential MMP disturbance and expressed a marked inhibitory effect on cell migration. Assessment of probable genetic toxicity by these extracts revealed no or minimum incidence of genetic toxicity. Therefore, the studied plant extracts are exhibiting potent anticancer activity based upon marked induction of tumor-cell death.

Keywords: cancer; Xylopia aethiopica; Imperata cylindrical; Echinops giganteus; Dorstenia psilurus; cell proliferation; metastasis

1. Introduction Cancer is the most serious disease worldwide that is expected to increase due to the adoption of behaviors and lifestyle factors known to cause cancer [1]. Toxicity and tumor resistance are currently the main limitations in using chemotherapeutic agents in cancer treatment. So, the discovery of new and safe treatment options is considered a big challenge [2]. Natural products represent a great source for screening new and safe anti-cancer products [3]. Plant extracts are rich in bioactive molecules based on the variety of their chemical constituents such as flavonoids, polyphenols and alkaloids which play a highly significant role in drug discovery and development process [4].

Pharmaceuticals 2020, 13, 357; doi:10.3390/ph13110357 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 357 2 of 19

As medicinal plants represent a good alternative, especially in developing countries, many of them are used in Africa for treatment of various diseases. Xylopia aethiopica (XA) which is a member of the family Annonaceae, is used as a spice in Western and Central Africa, as well as to treat bronchitis, headache and ulceration [5]. In addition to its anti-diabetic effect [6], anti-anaphylactic and anti-inflammatory activities [7], several studies have shown that XA extracts possess antibacterial and antifungal activities [8–11]. Imperata cylindrica (IC) (family Poaceae) also known as spear grass in West Africa, has diuretic, anti- inflammatory and antibacterial activities [12,13]. It also shows a potent anthelmintic activity [14] and the methanolic extract of its rhizomes was reported as a significant neuroprotective against glutamate-induced neurotoxicity in primary cultures of rat cortical cells [15]. Echinops giganteus (EG), (family Compositae) is traditionally used as a medicinal agent mainly in Africa and Asia. It is mainly used as heart and gastric troubles spice, reducing as well asthma attacks. In previous studies, the root methanolic extract showed significant antioxidant [16], antibacterial [17], and antifungal effects [18]. The methanolic extract of EG roots also exhibited a significant activity against M. tuberculosis [19], the methanolic extract from the underground part also reported for cytotoxic activity against prostate cancer (Mia PaCa2) and two leukemia cells (CCRF-CEM and CEM/ADR5000) [20]. Dorstenia psilurus (DP), (family Moraceae) is widely used in traditional medicine and represents a great source of active constituents including flavonoids, alkaloids and phenolic compounds [21–23]. It has a therapeutic effect on cardiovascular disorders, snakebites, headache and stomach disorders, moreover it exhibits a potent antimicrobial activity and as its methanolic showed antibacterial activity against a panel of Gram-negative bacteria including multidrug resistant (MDR) phenotypes [13]. Recent study reported the isolation of two isoprenylated flavones from the root extract of DP that activate AMP-activated protein kinase (AMPK), stimulate glucose uptake and lower glycemia [24]. Recently, various studies were conducted for screening the cytotoxic activity of these plant extracts against a variety of cancer types and resistance. These studies have shown a promising effect of using these extracts against some cancer cell lines [25–29]. Despite the useful biological activity expressed by certain plants, the study of their possible toxicity remains particularly important. As some chemicals or secondary metabolites from plants are toxins like substances that may cause harmful effects to humans. In this study, a well-established evaluation of the anticancer potential of these four plant extracts: (i) XA, (ii) IC, (iii) EG and (iv) DP was performed to assess their activities in the inhibition of cell proliferation in seven different human cancer cell lines: HeLa (cervical), MDA-MB-231 (breast), A549 (lung), HepG2 (liver), U-87 (glioblastoma, brain), SK-OV-3 (ovarian) and HL60 (leukemia). As well, an assessment of in vitro toxicity of these plant extracts was performed in non-cancerous HEK-293 cells. HeLa cells showed a higher sensitivity in cell proliferation assay upon treatment with these extracts, so further studies of the alteration and induction of cell death were investigated in HeLa cell line, by comparing the treated cells with these extracts to the untreated cells, using different assays involving cell cycle analysis, the caspase 3/7 activity and mitochondrial membrane potential (MMP). The effect of the studied medicinal plants on the inhibition of cell progression and metastasis was assessed using the wound healing assay. Furthermore, a single cell gel electrophoresis assay was performed to exclude any probable genetic toxicity upon using of these extracts in cancer treatment.

2. Results and Discussion

2.1. Cell Proliferation Assay The anti-proliferation effect is the first indication to be assessed when investigation novel antitumor agents, thus the cell growth inhibitory activity of the four plant extracts was initially assessed on the HeLa (cervical cancer) cell line 48 h after treatment with different concentrations of the crude methanol extracts. A dose dependent decrease in cell viability was observed (Figure1). At a concentration of 50 µg/mL of each crude extract, DP and EG inhibited the cell growth by more than 99%, followed by XA with > 90% and IC with > 80%. The comparative IC50 values of XA, IC, EG and DP for HeLa cells Pharmaceuticals 2020, 13, 357 3 of 19

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 19 were shown in (Figure1E). The most potent anti-proliferative e ffect was induced by DP with IC50 value ofwith 19.35 ICµ50g value/mL, followedof 19.35 µg/mL, by XA followed with IC 50byvalue XA with of 24.94 IC50 valueµg/mL. of 24.94 EG and µg/mL. IC showed EG and aIC quite showed similar a inhibitoryquite similar effect inhibitory on cell growtheffect on with cell growth IC50 values with ofIC 33.6550 values and of 38.14 33.65µ andg/mL, 38.14 respectively. µg/mL, respectively.

FigureFigure 1. 1.SRB SRB assayassay on HeLa HeLa cells cells to to evaluate evaluate the the effect eff ectof the of four the fourplant plant extracts: extracts: (A) Xylopia (A) Xylopia aethiopica aethiopica (XA), (XA),(B) Imperata (B) Imperata cylindrica cylindrica (IC), ((IC),C) Echinops (C) Echinops giganteus giganteus (EG) and (EG) (D) andDorstenia (D) Dorstenia psilurus (DP) psilurus on the (DP) cell on growth the cell growthafter 48 after h (X-axis: 48 h (X-axis: log concentrations log concentrations of extracts of extracts from 1 from to 200 1 to (µg/mL) 200 (µg /mL)and Y-axis: and Y-axis: the percentage the percentage of

ofnormalized normalized absorbance). absorbance). While While (E): (TheE): Thecomparison comparison of average of average IC50 of the IC50 fourof theplant four extracts plant over extracts 48 h (Y- over 48axis: h (Y-axis: concentration concentration in (µg/mL). in ( µValuesg/mL). are Values mean are± SD mean of threeSD independent of three independent experiments. experiments. ± OnOn the the other other hand, hand, after after 4848 hh treatmenttreatment of the non-tumor HEK293 HEK293 cells cells with with 50 50 µg/mLµg/mL of ofeach each crudecrude methanol methanol extract, extract, therethere waswas onlyonly aa negligiblenegligible effect effect on on the the cell cell growth. growth. The The cell cell viability viability was was 80.90%,80.90%, 73.88%, 73.88%, 71.89% 71.89% and and 70.26%70.26% afterafter cell treatment with with XA, XA, IC, IC, EG EG and and DP DP extracts, extracts, respectively. respectively. OnlyOnly for for concentrations concentrations above above 100100µ µg/mL,g/mL, aa decrease by more more than than 50% 50% in in cell cell viability viability could could be be seen seen (Figure(Figure2). 2). Upon Upon treatment treatment of of cells cells withwith aa concentrationconcentration of of 100 100 µg/mLµg/mL of of EG EG and and DP, DP, cell cell viability viability decreaseddecreased to to 49.16% 49.16% and and 49.44%, 49.44%, respectively. respectively. While Whil ate at a highera higher concentration concentration of of 200 200µg µg/mL/mL of of XA XA and ICand extracts, IC extracts, cell viability cell viability decreased decreased to 48.05% to 48.05% and and 47.30%, 47.30%, respectively. respectively. However However a a concentration concentration of belowof below 30 µ 30g/mL µg/mL could could be consideredbe considered safe safe for for healthy healthy cells cells with with cell cell viability viability more more than than 90%. 90%. These findings were in agreement with a recent study that was performed to assess the in vivo toxicological effect of the IC crude extract [30]. This study revealed the safety of crude methanol extract of IC at low doses on animal models, while prolonged use was suggested to be discouraged at high doses.

Figure 2. SRB assay on HEK293 cells to evaluate the effect of the four plant extracts: (A) XA, (B) IC, (C) EG and (D) DP on the cell growth after 48 h (X-axis: log concentrations of extracts from 1 to 200 (µg/mL) and Y-axis: the percentage of normalized absorbance). Values are mean ± SD of three independent experiments.

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 19 with IC50 value of 19.35 µg/mL, followed by XA with IC50 value of 24.94 µg/mL. EG and IC showed a quite similar inhibitory effect on cell growth with IC50 values of 33.65 and 38.14 µg/mL, respectively.

Figure 1. SRB assay on HeLa cells to evaluate the effect of the four plant extracts: (A) Xylopia aethiopica (XA), (B) Imperata cylindrica (IC), (C) Echinops giganteus (EG) and (D) Dorstenia psilurus (DP) on the cell growth after 48 h (X-axis: log concentrations of extracts from 1 to 200 (µg/mL) and Y-axis: the percentage of normalized absorbance). While (E): The comparison of average IC50 of the four plant extracts over 48 h (Y- axis: concentration in (µg/mL). Values are mean ± SD of three independent experiments.

On the other hand, after 48 h treatment of the non-tumor HEK293 cells with 50 µg/mL of each crude methanol extract, there was only a negligible effect on the cell growth. The cell viability was 80.90%, 73.88%, 71.89% and 70.26% after cell treatment with XA, IC, EG and DP extracts, respectively. Only for concentrations above 100 µg/mL, a decrease by more than 50% in cell viability could be seen (Figure 2). Upon treatment of cells with a concentration of 100 µg/mL of EG and DP, cell viability decreased to 49.16% and 49.44%, respectively. While at a higher concentration of 200 µg/mL of XA Pharmaceuticalsand IC extracts,2020 ,cell13, 357 viability decreased to 48.05% and 47.30%, respectively. However a concentration4 of 19 of below 30 µg/mL could be considered safe for healthy cells with cell viability more than 90%.

FigureFigure 2.2. SRBSRB assayassay onon HEK293HEK293 cellscells toto evaluateevaluate thethe eeffectffect ofof thethe fourfour plantplant extracts:extracts: (A) XA,XA, ((B)) IC,IC, ((CC)) EGEG andand (D) DP on on the the cell cell growth growth after after 48 48 h h(X-axis: (X-axis: log log concentrations concentrations of extracts of extracts from from 1 to 1 200 to 200(µg/mL) (µg/mL) and and Y-axis: Y-axis: the the percentage percentage of ofnormalized normalized absorbance). absorbance). Values Values are are mean mean ± SDSD of threethree ± independentindependent experiments.experiments.

2.2. Cytotoxicity of the Plant Extracts Screening of the cytotoxicity effect of the four studied plant extracts was performed using MTT viability assay. Seven cancer cell lines were incubated with the four extracts for 24, 48 and 72 h and the status of cell growth was observed (Figure3). The results showed that all of the aforementioned extracts expressed concentration dependent cytotoxic effects against the treated cell lines. Results confirmed that DP showed the most potent antitumor activity against cervical cancer (HeLa) cancer after 72 h, among the four studied extracts, with average half-maximal inhibitory concentration (IC50) values of 17.93, 21.61 and 28.27 µg/mL over 72, 48 and 24 h, respectively and 37.19 µg/mL. While IC expressed a higher cytotoxic activity against breast cancer (MDA-MB-231) after 72 h with IC50 of 27.30 µg/mL. EG expressed the most potent activity in growth inhibition of leukemia (HL60), while XA against glioblastoma brain cancer (U-87) with IC50 of 41.05 and 35.45 µg/mL, respectively. In lung cancer cells (A549), DP and XA were shown the most potent effect of growth inhibition after 72 h with IC50 of 39.96 and 40.79 µg/mL respectively. While the inhibitory effect of the studied plant extracts after 48 h against ovarian cancer (Sk-OV-3) cells was shown that DP and EG were more potent among the four extracts with IC50 value of 43.86, 44.38 µg/mL, respectively. The four extracts have possessed closely antitumor activity against hepatocellular carcinoma (HepG2) with a range from 32.99 to 53.21 µg/mL over 72 h. Pharmaceuticals 2020, 13, 357 5 of 19 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 5 of 19

Figure 3. 3. TheThe cytotoxicity cytotoxicity effect effect of ofthe the studied studied plant plant extracts extracts on different on different cell lines cell linesover over24, 48 24, and 48 72 and h using 72 h usingMTT assay: MTT assay:(A) HeLa, (A) HeLa, (B) HepG2, (B) HepG2, (C) MDA-MB-231, (C) MDA-MB-231, (D) A549, (D) A549, (E) SK-OV-3, (E) SK-OV-3, (F) U-87 (F) U-87 and and(G) (HL60,G) HL60, (Y- (Y-axis:axis: the theconcentration concentration of IC of50 ICin µg/mL).in µg/ mL).Values Values are mean are mean± SD of threeSD of independent three independent experiments. experiments. 50 ± In comparison comparison with with previous previous studies studies on ona variety a variety of Cameroonian of Cameroonian plant plant extracts extracts [25–29], [25 these–29], thesefindings findings confirm confirm the potential the potential anticancer anticancer activity activity of these of thesefour extracts four extracts in these in theseseven seven cancer cancer cell lines cell lineswith withconcentration concentration dependent dependent effect eff ectover over time. time. Ho However,wever, the the most most potent potent cytotoxic cytotoxic activity activity was against HeLa, HepG2 and MDA-MB-231 cells. According to the US National Cancer Institute (NCI) plant screening program, a crude plant extract is generally considered to have acceptable in vitro cytotoxiccytotoxic activityactivity ifif thethe ICIC5050 value is less than 20 µµg/mLg/mL after incubation 48 and 72 h treatment of cancer cell lines [31]. [31]. Based Based on on these these results, results, the four plantplant extractsextracts werewere shownshown potentpotent cytotoxiccytotoxic activityactivity againstagainst HeLaHeLa cellscells afterafter 7272 h,h, withwith ICIC5050 values of 17.93, 27.61, 30.10 and 32.29 µg/mL for DP, XA, EG and IC, respectively. Thus, HeLa cells were selected for the assessment of inhibition mechanisms.

Pharmaceuticals 2020, 13, 357 6 of 19 values of 17.93, 27.61, 30.10 and 32.29 µg/mL for DP, XA, EG and IC, respectively. Thus, HeLa cells were selected for the assessment of inhibition mechanisms. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 6 of 19 2.3. Live/Dead Staining 2.3. Live/Dead Staining A live/dead assay was performed to confirm the cytotoxic activity of the four plant extracts against A live/dead assay was performed to confirm the cytotoxic activity of the four plant extracts HeLa cell line. Cells were treated with a concentration of 30 µg/mL of each crude extract over 24, 48 against HeLa cell line. Cells were treated with a concentration of 30 µg/mL of each crude extract over and 72 h. Then, dual staining with fluorescence dyes was performed using calcein-AM and propidium 24, 48 and 72 h. Then, dual staining with fluorescence dyes was performed using calcein-AM and iodide. The results showed a significant decrease in viable cells after being with the extracts (Figure4A). propidium iodide. The results showed a significant decrease in viable cells after being with the Data confirmed the cytotoxic activity of these extracts with time dependent manners. As the percentage extracts (Figure 4A). Data confirmed the cytotoxic activity of these extracts with time dependent of viable cells of untreated cells was 97.10 0.8% after 72 h, while the percentage of viable cells manners. As the percentage of viable cells of± untreated cells was 97.10 ± 0.8% after 72 h, while the decreased to 75.36 5.5%, 60.36 4.3% and 49.46 3.8% after cell treatment with XA over 24, 48 and percentage of viable± cells decreased± to 75.36 ± 5.5%,± 60.36 ± 4.3% and 49.46 ± 3.8% after cell treatment 72 h, respectively. After treatment of cells with IC, viable cells decreased to 85 2.4%, 75.25 5.7% and with XA over 24, 48 and 72 h, respectively. After treatment of cells with IC, viable± cells decreased± to 56.79 4.9% over 24, 48 and 72 h, respectively. The percentage of viable cells dramatically decreased, 85 ± ±2.4%, 75.25 ± 5.7% and 56.79 ± 4.9% over 24, 48 and 72 h, respectively. The percentage of viable upon treatment of cells with EG to 59.77 5.9%, 48.28 3.1% and 41.35 2.4% over 24, 48 and 72 h, cells dramatically decreased, upon treatment± of cells with± EG to 59.77 ± 5.9%,± 48.28 ± 3.1% and 41.35 respectively.± 2.4% over 24, The 48 results and 72 confirmed h, respectively. that DP The was results shown confirmed a potent that cytotoxic DP was activity shown towards a potent HeLa cytotoxic cells, as the percentage of viable cells significantly decreased to 49.30 4.1%, 34.72 3.5% and 23.64 1.2% activity towards HeLa cells, as the percentage of viable cells significantly± decreased± to 49.30 ±± 4.1%, over34.72 24, ± 3.5% 48 and and 72 23.64 h, respectively ± 1.2% over (Figure24, 48 and4B–D). 72 h, These respectively results (Figures suggest 4B–D). that the These four results studied suggest plant extractsthat the are four potential studied cytotoxicplant extracts agents are for potentia the studiedl cytotoxic cell lines. agents for the studied cell lines.

FigureFigure 4. 4.A A livelive/dead/dead assayassay afterafter treatmenttreatment of HeLa cells with the four studied studied extracts extracts over over 24, 24, 48, 48, andand 72 72 h. h. ( A(A)) Green Green fluorescencefluorescence denotesdenotes viableviable cells stained with calcein-AM, calcein-AM, while while red red fluorescence fluorescence representsrepresents dead dead cells cells stained stained with with propidium propidium iodide.iodide. Then,Then, data analysis was was performed performed using using ImageJ ImageJ softwaresoftware and and Prism Prism 5 5 ( B(B)) 24 24 h, h, ( (CC)) 4848 hh andand ((DD)) 7272 h.h. All results are expresse expressedd as as a a total total percentage percentage of of viableviable and and dead dead cells cells from from four randomfour random fields withfields mean with standardmean ± standard deviation (SD)deviation of three (SD) independent of three ± determinations.independent determinations. Scale bar represents Scale bar 50 µrepresentsm. 50 µm.

2.4. Cell Cycle Analysis To investigate the mechanism of growth inhibition, the effect of the four studied crude extract on the cell cycle distribution of HeLa cells were analyzed after 48 h of the treatment of cells with the concentration of IC50 obtained from the MTT assay, followed by fixation and PI staining. The cell cycle arrest was analyzed using flow cytometry. The results in Figure 5 show an obvious alteration of the distribution of different phases. The cell population was seen to be increasingly accumulated

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2.4. Cell Cycle Analysis To investigate the mechanism of growth inhibition, the effect of the four studied crude extract on the cell cycle distribution of HeLa cells were analyzed after 48 h of the treatment of cells with the concentration of IC50 obtained from the MTT assay, followed by fixation and PI staining. The cell cycle arrest was analyzed using flow cytometry. The results in Figure5 show an obvious alteration of Pharmaceuticals 2020, 13, x FOR PEER REVIEW 7 of 19 the distribution of different phases. The cell population was seen to be increasingly accumulated at theat SubG0the SubG0/G1/G1 phase phase upon upon treatment treatment with with the studiedthe studied extracts. extracts. Compared Compared to the to untreatedthe untreated cells cells that showed a percentage of 6.02 1.94% in this phase, XA and IC showed a considerable increase with that showed a percentage of± 6.02 ± 1.94% in this phase, XA and IC showed a considerable increase percentages of 18.03 1.94% and 14.91 1.16%, respectively, while EG and DP significantly increased with percentages of± 18.03 ± 1.94% and± 14.91 ± 1.16%, respectively, while EG and DP significantly the accumulation of hypodiploid cells in the sub-G0/G1 phase with percentages of 38.88 2.84% and increased the accumulation of hypodiploid cells in the sub-G0/G1 phase with percentages± of 38.88 ± 44.512.84% and1.73%, 44.51 respectively. ± 1.73%, respectively. The data showed The data that showed there that was there no significant was no significant change in change cell arrest in cell in ± allarrest other in phases. all other The phases. results The suggest results that suggest HeLa th cellsat HeLa underwent cells underwent apoptosis apoptosis upon treatment upon treatment with the studiedwith the plant studied extracts. plant extracts.

FigureFigure 5. 5.Cell Cell cyclecycle analysisanalysis byby flowflow cytometrycytometry forfor HeLaHeLa cells treated with IC50 ofof the the different different studied studied plantplant extracts extracts for for 48 48 h: h: ((AA)) Control,Control, ((BB)) XA,XA, ((C)) IC, ( D) EG EG and and ( (EE)) DP. DP. While, While, ( (FF)) Comparative Comparative analysis analysis amongamong di differentfferent plant plant extracts extracts for for Sub-G0Sub-G0/G1,/G1, G0G0/G1,/G1, SS andand G2G2/M/M Phases.Phases. The cell cycle distribution distribution waswas analyzed analyzed by by ModFit ModFit LT LT software. software. AllAll resultsresults areare expressedexpressed in the histogram as as total total percentages percentages ofof cells cells from from four four di differentfferent groups groups with with mean mean ± SDSD ofof threethree independentindependent determinations.determinations. All All data data ± collectedcollected from from experiments experiments were were performed performed in three in th replicatesree replicates and analyzed and analyzed using theusing one-way the one-way analysis ofanalysis variance of (ANOVA)variance (ANOVA) at a significance at a significance level of p level< 0.05 of and p < indicated0.05 and indicated by **. by **.

PlantPlant extracts extracts exert exert their their cytotoxic cytotoxic eff ectseffects through through common common mechanisms mechanisms including including cell cycle cell cycle arrest andarrest cell and death cell by death apoptosis. by apoptosis. The potential The potential of anticancer of anticancer agents agents is evaluated is evaluated by the abilityby the toability initiate to cellinitiate cycle cell arrest cycle in arrest cancer in cells cancer [32 cells]. Production [32]. Production of apoptotic of apoptotic cells, whichcells, which are resulting are resulting from from DNA fragmentation,DNA fragmentation, display display a broad a hypo-diploid broad hypo-diploid sub-G0/ G1sub-G0/G1 peak which peak is which easily detectedis easily detected with suffi withcient losssufficient of cellular loss DNA of cellular and can DNA be discriminated and can be discriminated by flow cytometry by flow [33 cytometry]. Corresponding [33]. Corresponding to these findings, to inthese the present findings, study, in the the present four studied study, extracts the four have studie thed potential extracts have to cause the supotentialfficient DNAto cause loss sufficient to induce apoptosis,DNA loss as to the induce accumulation apoptosis, of as hypodiploid the accumulation cells in of the hypodiploid sub-G0/G1 cells phase in is the an sub-G0/G1 indication ofphase apoptotic is an cellindication death. Several of apoptotic intracellular cell death. cascades, Several such intracellu as the activationlar cascades, of caspases such as andthe theactivation disruption of caspases of MMP wouldand the be disruption a confirmation of MMP for apoptosis.would be a confirmation for apoptosis.

2.5. Annexin V-FITC/PI for Apoptosis Detection In order to verify that the effect of the studied extracts on the growth inhibition of HeLa cells was related to apoptosis, analysis of the apoptotic and necrotic cells was performed using Annexin V. After 48 h of the treatment with the IC50 of the four extracts, a double labelling was done with PI that staining the necrotic cells with red fluorescence and Annexin V-FITC which produce cytoplasmic green labelling for apoptotic cells. Images from fluorescent microscopy (Figure 6A), showed that viable cells were negative for both PI and Annexin V. While XA and IC were shown considerable

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2.5. Annexin V-FITC/PI for Apoptosis Detection In order to verify that the effect of the studied extracts on the growth inhibition of HeLa cells was related to apoptosis, analysis of the apoptotic and necrotic cells was performed using Annexin V. After 48 h of the treatment with the IC50 of the four extracts, a double labelling was done with PI that staining the necrotic cells with red fluorescence and Annexin V-FITC which produce cytoplasmic green labelling for apoptotic cells. Images from fluorescent microscopy (Figure6A), showed that viable cells were negativePharmaceuticals for 2020 both, 13, PIx FOR and PEER Annexin REVIEW V. While XA and IC were shown considerable green 8 of and19 red labellinggreen for and both red apoptotic labelling for and both necrotic apoptotic cells, and respectively. necrotic cells, Arespectively. significant A detection significant of detection apoptotic of cells was shownapoptotic upon cells cell was treatment shown upon with bothcell treatment EG and DP,with indicating both EG and that DP, cells indicating mainly underwentthat cells mainly apoptosis with thoseunderwent extracts. apoptosis with those extracts.

FigureFigure 6. Detection 6. Detection of of apoptosis apoptosis and necr necrosisosis using using Annexin Annexin V-FITC V-FITC and PI anddual PIstaining dual after staining 48 h after of treatment of HeLa cells with IC50 of the studied crude extracts XA, IC, EG and DP in comparison 48 h of treatment of HeLa cells with IC50 of the studied crude extracts XA, IC, EG and DP in comparisonto untreated to untreated cells. (A) cells.Images ( Aby) Imagesfluorescent by microscopy fluorescent represent microscopy one of represent the three independent one of the three experiments, (B) Results, after flow cytometry analysis, are expressed in the histogram as total independent experiments, (B) Results, after flow cytometry analysis, are expressed in the histogram percentages of cells from four different quadrants (Q1 = early apoptosis, Q2 = late apoptosis, Q3 = as totalnecrosis, percentages Q4 = live), of cells with from mean four ± SD di offf erentthree independent quadrants (Q1determinations.= early apoptosis, All results Q2 are= expressedlate apoptosis, Q3 = necrosis, Q4 = live), with mean SD of three independent determinations. All results are in the histogram as total percentages± of cells from four different groups with mean ± SD of three expressedindependent in the histogram determinations. as total (C) percentagesBar graph for of values cells fromof cell four percentages, different expressed groups with in mean mean ± SD ± of threestandard independent deviation determinations. (SD) and analyzed (C )using Bar graphthe one-way for values analysis of of cell variance percentages, (ANOVA) expressed at a in mean significancestandard level deviation of p < 0.05 (SD) and and indicated analyzed by **. using the one-way analysis of variance (ANOVA) at a ± significance level of p < 0.05 and indicated by **. The visual observations were confirmed by the quantitative analysis by flow cytometry. Data in TheFigures visual 6B–C observations shows the weredistribution confirmed of HeLa by thecells quantitative within four analysisdifferent byquadrants flow cytometry. (Q1 = early Data in Figureapoptosis,6B–C shows Q2 = the late distribution apoptosis, Q3 of = HeLanecrosis, cells Q4 within = live) and four represent di fferent one quadrants of three sets (Q1 of =independentearly apoptosis, experiments conducted. Cells that are undergoing apoptosis will shift from the viable quadrant (Q4) to the early apoptosis quadrant (Q1), and eventually end up in late apoptosis quadrant (Q2). On the other hand, cells that undergo necrosis will shift from viable quadrant (Q4) to the late necrosis quadrant (Q3). Un-treated cells showed a percentage of 92.8 ± 1.06% for viable cells, 4.06 ± 0.73% for

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Q2 = late apoptosis, Q3 = necrosis, Q4 = live) and represent one of three sets of independent experiments conducted. Cells that are undergoing apoptosis will shift from the viable quadrant (Q4) to the early apoptosis quadrant (Q1), and eventually end up in late apoptosis quadrant (Q2). On the other hand, cellsPharmaceuticals that undergo 2020, 13 necrosis, x FOR PEER will REVIEW shift from viable quadrant (Q4) to the late necrosis quadrant 9 (Q3). of 19 Un-treated cells showed a percentage of 92.8 1.06% for viable cells, 4.06 0.73% for dead cells, ± ± 2.11dead cells,0.30% 2.11for ± late 0.30% apoptosis for late and apoptosis 1.04 0.09%and 1.04 for ± early 0.09% apoptosis. for early XAapoptosis. and IC expressedXA and IC increase expressed in ± ± theincrease late apoptotic in the late population apoptotic topopulation 18 2.08% toand 18 ± 7.05 2.08%2.6%, and 7.05 respectively. ± 2.6%, respectively. While EG and While DP showedEG and ± ± significantDP showed increase significant in the increase late apoptosis in the late to apoptosis 31.50 1.26% to 31.50 and ± 35.6 1.26%1.42%, and 35.6 respectively. ± 1.42%, respectively. ± ± AsAs well,well, aa considerableconsiderable increaseincrease waswas shownshown inin necroticnecrotic cells,cells, withwith percentagespercentages ofof 15.3015.30 ± 2.35%, ± 22.4022.40 ± 3.82%, 16.90 ± 3.36%3.36% and and 25.50 25.50 ± 4.54%,4.54%, upon upon treatment treatment with with XA, XA, IC, IC, EG EG and and DP, DP, respectively. respectively. ± ± ± Lastly,Lastly, earlyearly apoptotic cells, represented represented by by Q1, Q1, displa displayedyed only only a aslight slight increase increase in incell cell distribution distribution as asa result a result of oftreatment treatment with with the the studied studied extracts extracts XA, XA, IC, IC,EG EGand and DP DPto percentages to percentages of 2.51 of 2.51± 0.34%,0.34%, 5.99 ± 5.99± 1.36%,1.36% 7.05, 7.05± 1.23%,1.23%, 4.11 4.11± 0.89%,0.89%, respectively. respectively. Phosphatidyl-s Phosphatidyl-serineerine (PS) (PS)on the on outer the outer layer layer of the of ± ± ± theplasma plasma membrane membrane acts acts as asa recognition a recognition site site of ofphagocytes phagocytes during during the the early early stage stage of of apoptosis apoptosis [34]. [34]. AnnexinAnnexin V,V, aa calcium-dependentcalcium-dependent proteinprotein cancan bindbind toto thethe exposedexposed phosphatidyl-serinephosphatidyl-serine (PS)(PS) onon thethe externalexternal layerlayer ofof thethe membranemembrane [[35].35]. In In this stud study,y, it was observed that thethe percentagepercentage ofof cellscells undergoingundergoing latelate apoptosis apoptosis increased increased significantly, significantly, thus thus confirming confirming that that apoptosis apoptosis was onewas of one the of major the modesmajor modes of cell deathof cell induceddeath induced by the fourby the studied four studie plantd extracts, plant extracts, especially especially EG and EG DP and extracts. DP extracts.

2.6.2.6. EEffectffect on the Activity ofof CaspaseCaspase 33/7/7 ToTo investigateinvestigate whether whether the the apoptosis apoptosis effect effect induced induced by the fourby the studied four plantstudied extracts plant is dependentextracts is upondependent the caspases upon the activation, caspases activation, the caspase the 3caspase/7 activity 3/7 activity examined examined on HeLa on HeLa cells treatedcells treated with with the concentrationthe concentration of IC 50of valuesIC50 values of the of four the studied four studied extracts extracts for 24 h (Figurefor 24 h7). (Figure DP expressed 7). DP aexpressed significant a esignificantffect on activation effect on ofactivation caspase 3of/7 caspase up to 7.17 3/7 up0.72 to 7.17 fold ± compared0.72 fold compared to the untreated to the untreated cells. As well,cells. ± EGAs ewell,fficiently EG efficiently enhanced enhanced the caspase the 3/ 7caspase activity 3/7 by activi 5.15 ty1.14 by 5.15 fold, ± while 1.14 fold, cells while treated cells with treated XA and with IC ± expressedXA and IC an expressed increase in an the increase activities in ofthe caspase activities 3/7 byof 3.60caspase0.58 3/7 and by 4.753.60 ± 0.920.58 foldand compared4.75 ± 0.92 to fold the ± ± untreatedcompared cellsto the that untreated expressed cells only that 1.12 expressed0.19 fold only increase 1.12 ± 0.19 in activity. fold increase in activity. ±

FigureFigure 7.7. Enzymatic activity of caspase 33/7/7 onon HeLlaHeLla cellscells andand afterafter foldfold increaseincrease inin activityactivity afterafter 2424 hh treatment of with IC50 of the four studied plant extracts XA, IC, EG and DP. All data expressed as treatment of with IC50 of the four studied plant extracts XA, IC, EG and DP. All data expressed as mean meanstandard ± standard deviation deviation (SD) at (SD) a significance at a significance level of levelp < 0.05of p < and 0.05 indicated and indicated by **. by **. ± OneOne ofof thethe important measures thatthat play essential roles in apoptosis, necrosis, and inflammationinflammation isis caspasescaspases whichwhich areare aa familyfamily ofof cysteinecysteine proteases,proteases, causing cleavage of cellular protein [36]. [36]. ExaminationExamination ofof the the activities activities of of caspase caspase 3/7 3/7 in HeLa in HeLa cells cells with with the four the studiedfour studied plant extractsplant extracts has shown has ashown marked a marked increase increase in the activity in the ofactivity caspase of 3caspase/7, which 3/7, confirm which theconfirm effect the of these effect extracts of these in extracts apoptotic in cellapoptotic death ascell observed death as previouslyobserved previously in cell cycle in analysis cell cycle and analysis Annexin and V. Annexin V.

2.7.2.7. EEffectffect on MitochondrialMitochondrial MembraneMembrane PotentialPotential (MMP)(MMP) AsAs thethe disruptiondisruption ofof thethe MMPMMP isis oneone ofof thethe sequentialsequential eventsevents exhibitedexhibited duringduring thethe apoptoticapoptotic pathway,pathway, thethe eeffectffect ofof thethe fourfour studiedstudied plantplant extractsextracts inin thisthis alterationalteration afterafter 2424 hh ofof treatmenttreatment againstagainst HeLa cells was compared to the positive control FCCP (Figure 8A) with untreated cells representing the negative control. After the cells were treated with IC50 of XA and EG, the MMP closely dropped to 19.70% and 20.84%, respectively. As well, DP and IC disrupted the MMP to 23.02% and 26.90%, while FCCP, the positive control for the MMP breakdown, altered the MMP to 30.20% compared to the control cells which represent the 100%. The results were analyzed in Figure 8B to assess the fold change in MMP after the treatment of different studied plant extracts. The data showed that all plant

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HeLa cells was compared to the positive control FCCP (Figure8A) with untreated cells representing the negative control. After the cells were treated with IC50 of XA and EG, the MMP closely dropped to 19.70% and 20.84%, respectively. As well, DP and IC disrupted the MMP to 23.02% and 26.90%, while FCCP, the positive control for the MMP breakdown, altered the MMP to 30.20% compared to the control cells which represent the 100%. The results were analyzed in Figure8B to assess the fold change in MMP after the treatment of different studied plant extracts. The data showed that all plant Pharmaceuticals 2020, 13, x FOR PEER REVIEW 10 of 19 extracts expressed a significant alteration of the MMP. XA revealed a significant alteration decrease by 5.09 0.21-fold, followed by EG, DP and IC with 4.80 0.12, 4.37 0.43 and 3.77 0.63-fold, extracts± expressed a significant alteration of the MMP. XA revealed± a significant± alteration decrease± respectively.by 5.09 ± Comparatively, 0.21-fold, followed FCCP by alteredEG, DP theand MMPIC with with 4.80 a 3.35-fold± 0.12, 4.37 decrease. ± 0.43 and Further 3.77 ± analysis0.63-fold, using fluorescencerespectively. microscopy Comparatively, (Figure FCCP8C) was altered performed the MMP to with visualize a 3.35-fold the breakdown decrease. Further of MPP analysis after using treatment of HeLafluorescence cells with microscopy the IC50 of(Figure the four 8C) studiedwas perfor plantmed extracts, to visualize followed the breakdown by staining of withMPP 500after nM of tetramethylrhodaminetreatment of HeLa cells ethyl with ester the IC (TMRE.)50 of the four The studied results plant represent extracts, the followed significant by staining depletion with of500 MMP afternM cells of weretetramethylrhodamine treated with the ethyl four ester plant (TMRE.) extracts, The comparedresults represent to the the positive significant control depletion (FCCP) of and MMP after cells were treated with the four plant extracts, compared to the positive control (FCCP) the untreated cells, as the remaining living cells obviously showed a depolarization of MMP which and the untreated cells, as the remaining living cells obviously showed a depolarization of MMP induced the apoptotic pathway and cell death. which induced the apoptotic pathway and cell death.

FigureFigure 8. E ff8.ect Effect of the of the IC 50ICvalues50 values of of the thefour four plant extracts extracts an andd 50 50 µMµM positive positive control control (FCCP) (FCCP) on the on the MMPMMP of HeLa of HeLa cells cells after after 24h. 24h. Un-treated Un-treated cellscells representingrepresenting the the negative negative control. control. (A) (FluorescenceA) Fluorescence IntensityIntensity after after 24 h treatment,24 h treatment, (B) Analysis (B) Analysis of fold of Changefold Change of MMP. of MMP. Values Values expressed expressed in mean in meanstandard ± ± deviationstandard (SD). deviation (C) Fluorescence (SD). (C) Fluorescence Images after Images 24 h after treatment, 24 h treatment, visualize visualize the breakdown the breakdown of MPP of after MPP after treatment of HeLa cells with the IC50 of the four studied plant extracts, followed by staining treatment of HeLa cells with the IC50 of the four studied plant extracts, followed by staining by 500 nM by 500 nM of TMRE. of TMRE. Mitochondria play an essential role in the physiological metabolism of cells and energy supply Mitochondria play an essential role in the physiological metabolism of cells and energy supply for cell survival [37]. Mitochondrial membrane potential (MMP) is one of the sequence processes that for cellintegrated survival during [37]. apoptotic Mitochondrial pathway. membrane Results in potential this study (MMP) revealed is onethat ofthe the four sequence studied plant processes that integratedextracts showed during significant apoptotic disruption pathway. of Results the MMP in thiscompared study revealedto the untreated. that the These four studiedfindings plant extractsconfirm showed that significantcells undergo disruption apoptosis ofafter the treatment MMP compared with these to extracts the untreated. by potential These MMP findings alteration confirm that cellswhich undergo represent apoptosis is a key step after in the treatment intrinsic withapoptotic these pathway extracts by potential MMP alteration which represent is a key step in the intrinsic apoptotic pathway 2.8. Wound Healing Assay Assessment of the inhibitory effect of the four studied plant extracts on the progression and migration was performed against HeLa cells over 24 and 48 h. the negative control is represented by un-treated cells and cells treated with vehicle (0.1% DMSO). The treatment of cells with the IC50 of the studied plant extracts resulted in a significant blocking the progression and wound healing of the

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2.8. Wound Healing Assay Assessment of the inhibitory effect of the four studied plant extracts on the progression and migration was performed against HeLa cells over 24 and 48 h. the negative control is represented by un-treated cells and cells treated with vehicle (0.1% DMSO). The treatment of cells with the IC50 of the studied plant extracts resulted in a significant blocking the progression and wound healing of the Pharmaceuticals 2020, 13, x FOR PEER REVIEW 11 of 19 scratch area compared to the untreated cells and control cells treated with the solvent (0.1% DMSO) as a controlscratch (Figurearea compared9A). Data to the analysis untreated of cells the percentageand control cells of the treated wound with closure the solvent was (0.1% performed DMSO) and data showedas a control that (Figure the percentage 9A). Data decreasedanalysis of the at 24 percentage h from 96% of the and wound 81% forclosure untreated was performed cells and and DMSO, respectivelydata showed to 38%, that 36%, the percentage 27% and decreased 11.5% for at IC, 24 XA,h from EG 96% and and DP. 81% After for untreated 36 h, the cells scratch and areaDMSO, for the untreatedrespectively cells was to 38%, totally 36%, covered 27% and by 11.5% cellswith for IC, a percentageXA, EG and ofDP. 100% After and 36 h, for the cells scratch treated area with for the DMSO by 95%.untreated However, cells the was significant totally covered effect by on cells the scratchwith a percentage area, which of continued 100% and upfor tocells 36 h,treated was observedwith in cellsDMSO treated by 95%. with However, DP where the the significant percentage effect of on wound the scratch closure area, was which only continued 16.5% followedup to 36 h, by was 39.5% observed in cells treated with DP where the percentage of wound closure was only 16.5% followed for cells treated with EG. While the coverage area of the scratch increased to 64.5% and 66% for cells by 39.5% for cells treated with EG. While the coverage area of the scratch increased to 64.5% and 66% treated with IC and XA, respectively (Figure9B), from these findings it was obvious that the four for cells treated with IC and XA, respectively (Figure 9B), from these findings it was obvious that the studiedfour plant studied extracts plant exhibitedextracts exhibited a potential a potential inhibitory inhibitory effect effect of metastatic of metastatic progression. progression.

FigureFigure 9. Eff 9.ect Effect of the of the plant plant extracts extracts on on cell cell growth; growth; ScratchingScratching was was performed performed using using a 200 a 200µL pipetteµL pipette tip. (A) Metastatic progression by scratch test. While, (B) Time dependent cell wound closure % under tip. (A) Metastatic progression by scratch test. While, (B) Time dependent cell wound closure % under the influence of the four plant extracts XA, IC, EG and DP compared to control (un-treated cells) and the influence of the four plant extracts XA, IC, EG and DP compared to control (un-treated cells) and cells treated with vehicle (0.1% DMSO) as negative control. Values expressed in mean ± standard cells treated with vehicle (0.1% DMSO) as negative control. Values expressed in mean standard deviation (SD). ± deviation (SD). Cell migration in vitro, or cell metastasis in vivo, represents one of the main features of Cellmalignant migration tumorsin vitrothat ,leads or cell to metastasisincrease in inthe vivo mort, representsality rate in one cancer of the disease main [38]. features In this of study, malignant tumorswound-healing that leads to assay increase showed in the that mortality the four rate studied in cancer plant disease extracts [ 38significantly]. In this study, inhibited wound-healing the cell assaymigration showed thatin HeLa the cells four when studied treated plant with extracts the IC significantly50 values. DP and inhibited EG showed the cell more migration potential inand HeLa cells whencontinuous treated effect with in the the inhibition IC50 values. of wound DP and closure EG up showed to 36 h. more These potential findings could and continuousrepresent potent effect in the inhibitionanticancer of agents wound that closure inhibited up cancer to 36 metastasis. h. These findings could represent potent anticancer agents that inhibited cancer metastasis. 2.9. Single Cell Gel Electrophoresis Assay To assess if there is any possible toxicity induced by the studied plant extracts especially damage to the DNA and genotoxicity, the comet assay was performed. The principle of the comet assay is based on the fact that DNA strands which occur as a negatively charged supercoiled structure in the nucleus can be fragmented due to the exposure to the toxins or drug treatments [39].

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2.9. Single Cell Gel Electrophoresis Assay To assess if there is any possible toxicity induced by the studied plant extracts especially damage to the DNA and genotoxicity, the comet assay was performed. The principle of the comet assay is based on the fact that DNA strands which occur as a negatively charged supercoiled structure in the nucleus can be fragmented due to the exposure to the toxins or drug treatments [39]. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 12 of 19 According to Figure 10, it can be observed that no direct DNA strand breakage was caused by IC50 levelsAccording of the three to Figure of the 10, studied it can plantbe observed extracts—IC, that no direct EG and DNA DP—compared strand breakage to was the untreatedcaused by cells and vehicleIC50 levels control of the (0.1%three of DMSO). the studied While plant IC extrac50 ofts—IC, the plant EG and extract DP—compared XA might interfereto the untreated with low cells DNA damage.and vehicle Several control parameters (0.1% DMSO). including While percentage IC50 of the of plant DNA extract in comet XA tailmight (% interfere tail-DNA), with tail low length DNA (TL), and taildamage. moment Several (TM) parameters were used including in the percentage past to monitor of DNA DNA in comet strand-breakage tail (% tail-DNA), with tail thelength comet (TL), assay. In thisand study, tail moment the percentage (TM) were of used tail-DNA in the past was to usedmonitor to quantifyDNA strand-breakage DNA strand-breakage with the comet in assay. Hela cells afterIn 24 this h of study, treatment the percentage with the plantof tail-DNA extracts. was Data used in to Table quantify1, confirmed DNA strand-breakage the results as in IC, Hela EG cells and DP after 24 h of treatment with the plant extracts. Data in Table 1, confirmed the results as IC, EG and exhibited percentage (% tail-DNA) <10% which represent the value for undamaged nuclei, while XA DP exhibited percentage (% tail-DNA) <10% which represent the value for undamaged nuclei, while shown value (% tail-DNA) of 19.04 2.10% that indicate low-damaged nuclei [40]. From these results, XA shown value (% tail-DNA) of± 19.04 ± 2.10% that indicate low-damaged nuclei [40]. From these it canresults, be assumed it can be that assumed these plant that these extracts plant can extracts be used can inbe in-vivo used in studiesin-vivo studies with no with or minimumno or minimum incidence of geneticincidence toxicity. of genetic toxicity.

FigureFigure 10. Comet 10. Comet assay: assay: fluorescence fluorescence micrographs micrographs of genotoxicity genotoxicity to toHela Hela cells cells obtained obtained after after24 h of 24 h of the treatmentthe treatment of the of the four four studied studied plant plant extracts extracts XA,XA, IC, EG EG and and DP DP compared compared to untreated to untreated cells cellsand and vehiclevehicle (0.1 DMSO)(0.1 DMSO) treated treated cells. cells.

TableTable 1. Percentage 1. Percentage of theof the head head and and tail tail of of DNA DNA afterafter cell treatment treatment with with IC IC50 of50 studiedof studied extracts. extracts.

Head Head DNA DNA (%) (%) ±SD SD Tail DNA (%) (%) Control 99.78 ± 0.07± 0.21 Control 99.78 0.07 0.21 DMSO 94.63± ± 1.09 5.36 DMSO 94.63 1.09 5.36 Xylopia aethiopica (XA) 80.96± ± 2.10 19.04 Xylopia aethiopica (XA) 80.96 2.10 19.04 Imperata cylindrica (IC) 91.82± ± 1.34 8.17 ImperataEchinops cylindrica giganteus(IC) (EG) 91.82 96.49 1.34± 0.79 8.17 3.5 ± EchinopsDorstenia giganteus psilurus(EG) (DP) 96.49 93.11 0.79± 1.33 6.89 3.5 ± Dorstenia psilurus (DP) 93.11 1.33 6.89 ± 3. Materials and Methods 3. Materials and Methods 3.1. Materials 3.1. Materials3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), tetramethylrhodamine ethyl 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazoliumester (TMRE), sulphorhodamine B (SRB), trichloroacetic bromide acid (MTT), (TCA), tetramethylrhodamine Tris base (tris [hydroxymethyl]aminomethane), calcin AM, annexin V and propidium iodide (PI) were obtained from ethyl ester (TMRE), sulphorhodamine B (SRB), trichloroacetic acid (TCA), Tris base Sigma Aldrich Chemie GmbH (Taufkirchen, Germany). Dimethyl sulfoxide (DMSO) was procured (tris [hydroxymethyl]aminomethane), calcin AM, annexin V and propidium iodide (PI) were obtained from Carl Roth GmbH & Co. (Karlsruhe, Germany). Roswell Park Memorial Institute (RPMI) 1640 fromMedium, Sigma Aldrich Dulbecco’s Chemie modified GmbH Eagle’s (Taufkirchen, minimum Germany).essential medium Dimethyl (DMEM), sulfoxide Fetal bovine (DMSO) serum was (FBS) procured fromwere Carl purchased Roth GmbH from & Capricon Co. (Karlsruhe, Scientific (Ebsdorfergrund, Germany). Roswell Germany) Park. MemorialThe Caspase-Glo Institute 3/7 Assay (RPMI) kit 1640 was procured from Promega (Walldorf, Germany), and carbonyl cyaninde 4-(trifluoromethoxy) phenylhydrazone (FCCP) was from Cayman Chemical (Ann Arbor, Michigan, USA).

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Medium, Dulbecco’s modified Eagle’s minimum essential medium (DMEM), Fetal bovine serum (FBS) were purchased from Capricon Scientific (Ebsdorfergrund, Germany). The Caspase-Glo 3/7 Assay kit was procured from Promega (Walldorf, Germany), and carbonyl cyaninde 4-(trifluoromethoxy) phenylhydrazonePharmaceuticals 2020, 13 (FCCP), x FOR wasPEERfrom REVIEW Cayman Chemical (Ann Arbor, Michigan, USA). 13 of 19 3.2. Plant Material and Extraction 3.2. Plant Material and Extraction Extraction of the four studied plants: Xylopia aethiopica (XA), Imperata cylindrica (IC), Echinops Extraction of the four studied plants: Xylopia aethiopica (XA), Imperata cylindrica (IC), Echinops giganteus (EG) and Dorstenia psilurus (DP) (collected in Cameroon) were performed at our partner giganteus (EG) and Dorstenia psilurus (DP) (collected in Cameroon) were performed at our partner laboratory at the University of Dschang, Cameroon. Plant materials were purchased from Dschang laboratory at the University of Dschang, Cameroon. Plant materials were purchased from Dschang local local market, West Region of Cameroon in August 2018. They were identified at the Cameroonian market, West Region of Cameroon in August 2018. They were identified at the Cameroonian National National Herbarium where voucher specimens were deposited under the flowing reference number Herbarium where voucher specimens were deposited under the flowing reference number (Table 2, (Table2, Figure 11). The extraction was done by maceration of 100 g of the plant material in 500 mL Figure 11). The extraction was done by maceration of 100 g of the plant material in 500 mL methanol methanol for 48 h, then, the methanolic extracts were concentrated by rotary evaporation under reduced for 48 h, then, the methanolic extracts were concentrated by rotary evaporation under reduced pressure pressure to obtain the crude extracts. The extracts were then conserved at 4 C until further use. to obtain the crude extracts. The extracts were then conserved at 4 °C until further◦ use.

Table 2. The botanical origin of the four studied plant extracts. Table 2. The botanical origin of the four studied plant extracts.

ScientificScientific Name Name Family Family Parts Parts Used Used Voucher Voucher Numbe Numberr Xylopia aethiopicaXylopia aethiopica (Dunal) (Dunal) A. Rich A. Rich AnnonaceaeAnnonaceae fruits fruits 16419/SRF-Cam 16419/SRF-Cam ImperataImperata cylindrica cylindrica(L.) Raeusch. (L.) Raeusch. Poaceae Poaceae roots roots 30139/SRFK 30139/SRFK EchinopsEchinops giganteus giganteusA. Rich. A. Rich. Compositae Compositae roots roots 23647/SRF-Cam 23647/SRF-Cam DorsteniaDorstenia psilurus psilurusWelwitch Welwitch Moraceae Moraceae roots roots 44839/HNC 44839/HNC

FigureFigure 11. 11.Morphological Morphological characteristics characteristics of of the the studied studied plants: plants: ( A(A)) XA, XA, ( B(B)) IC, IC, ( C(C)) EG EG and and ( D(D)) DP. DP.

3.3.3.3. CellCell Lines Lines and and Cell Cell Culture Culture TheThe HEK293 HEK293 cell cell line line (human (human embryonic embryonic kidney) kidney) and the and human the cancerhuman cell cancer lines: cell HeLa lines: (cervical), HeLa MDA-MB-231(cervical), MDA-MB-231 (breast), A549 (breast), (lung), A549 HepG2 (lung), (liver), HepG U-872 (liver), (glioblastoma, U-87 (glioblastoma, brain), SK-OV-3 brain), (ovarian) SK-OV-3 and(ovarian) HL60 (acuteand HL60 promyelocytic (acute promyelocytic leukemia) wereleukemia) obtained were from obtained the American from the Type American Culture Type Collection Culture (ATCC,Collection Manassas, (ATCC, VA, Manassas, USA). The VA, cells USA). were The grown cells in were recommended grown in culturerecommended media supplemented culture media TM withsupplemented 10% FBS and with MEM-non-essential 10% FBS and MEM-non-essential amino- acids (Gibco amino-Thermo-Fisher, acids (Gibco Waltham,TM Thermo-Fisher, MA, USA), 50Waltham,µg/mL gentamicin MA, USA), and 50 µg/mL 2.5 µg/ gentamicinmL amphotericin and 2.5 B µg/mL in a 5–7% amphotericin CO2 humidified B in a 5–7% atmosphere CO2 humidified at 37 ◦C. Theatmosphere cells were at grown 37 °C. asThe a monolayercells were grown for no moreas a monolayer than 15 passages. for no more than 15 passages.

3.4. SRB Assay To initially assess the possible cytotoxic effect of the studied plant extracts, the colorimetric sulphorhodamine-B (SRB) assay was used for measurement of cell proliferation [41]. Briefly, 4 × 104 of HeLa (cervical cancer) and HEK293 (non-tumor) cells were added to each well of a 96-well plate and incubated overnight to allow for cell attachment. The cells were then treated with serial dilutions of the four plant extracts: XA, IC, EG and DP (200 to 1 µg/mL) and 1% Triton-x was used as a positive

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3.4. SRB Assay To initially assess the possible cytotoxic effect of the studied plant extracts, the colorimetric sulphorhodamine-B (SRB) assay was used for measurement of cell proliferation [41]. Briefly, 4 104 of × HeLa (cervical cancer) and HEK293 (non-tumor) cells were added to each well of a 96-well plate and incubated overnight to allow for cell attachment. The cells were then treated with serial dilutions of the four plant extracts: XA, IC, EG and DP (200 to 1 µg/mL) and 1% Triton-x was used as a positive control. Untreated cells receiving the same volume of medium were served as a control while the concentration of vehicle control (DMSO) was kept at or below 0.1%. After 48 h exposure, the cells were fixed with ice-cold 10% TCA at 4 ◦C for 1 h, then washed four times under slow-running tap water, afterwards, stained with 0.057% (w/v) SRB in 1% acetic acid, washed and air-dried. Bound dye was solubilized with 200 µL of 10 mM Tris base solution (pH 10.5). The plates were read at 540 nm absorbance on a Fluostar microplate reader (BMG Labtech, Ortenberg, Germany), the determination of 50% inhibitory concentration (IC50) was based on dose-response curves between the extract concentration and percent growth inhibition using the GraphPad Prism 5 software. The values are expressed as mean SD with ± all the experiments independently performed in triplicate.

3.5. Cell Viability Assay Cell viability was measured by a standard MTT assay method [42]. The plant extracts were dissolved in DMSO, then diluted with cell culture medium to different serial dilutions (200 to 1 µg/mL) while the concentration of vehicle control (DMSO) was kept at or below 0.1%., 1% Triton-x was used as a positive control and untreated cells receiving the same volume of medium served as control For all adherent cells, the cells were cultured in 96-well plates (4 104 cells per well) and incubated × 1 at 37 ◦C for 24, 48 and 72 h. After that, a 20 µL MTT (5 mg mL− ) solution was added to each well and incubated for 4 h, then 150 µL of DMSO was added to each well to dissolve the formazan crystals. The absorbance data was detected by a microplate reader at 490 nm. For Leukemia HL60, a suspension of 4 104 cells/mL were seeded in 96-well plates and immediately after, serial dilution of × the extracts in the media was added. After adequate incubation 24, 48 and 72 h, the 96-well plates were centrifuged at 1000 g, 4 C for 5 min in a microplate-compatible centrifuge, then the media was × ◦ carefully aspirated and treated with MTT solution as above mentioned. The viability was observed based on comparison with the absorbance of untreated and treated cells. The IC50 values were obtained from the concentration of the plant extracts that induced 50% inhibition of cell growth using the GraphPad prism 5 software. Additionally, each test was replicated three times independently.

3.6. Live Dead Staining Assessment of cell viability of HeLa cells upon treatment with the four studied extracts was conducted using the LIVE/DEAD® Viability/Cytotoxicity Kit. A total of 2.0 105 cells/well were × plated on the surface of a sterile glass coverslip placed in a twelve-well plate and incubated overnight before treatment with a concentration of 30 µg/mL of the crude extracts. Cells were washed with 1 × PBS solution before staining using a dual-fluorescence of calcein-AM (2.0 µM) and propidium iodide (20 µg/mL). Cell washed twice after 15 min with 1 PBS solution, then visualization of samples was × carried out using a fluorescence microscope (CKX-53 Olympus, Tokyo, Japan). Viable cells stained with green fluorescence and dead cells labelled with red fluorescence correlating to calcein-AM and Propidium iodide, respectively. Four random fields of view for each sample were captured and were analyzed using ImageJ software for three independent experiments. The percentages of viable cells were calculated as follows:

Viable cells (%) = [live cells/(live cells + dead cells)] 100 (1) × Pharmaceuticals 2020, 13, 357 15 of 19

3.7. Cell Cycle Analysis A total of 3 105 cells were seeded into a well of a 12-well plate and incubated with the IC values × 50 obtained from the MTT assay of the four studied plant extracts and untreated cells as a control for 24 h. Following incubation, cells were trypsinized, washed with PBS and resuspended in 100 µL of ice-cold PBS. Cells transferred to a 2 mL sample tube and shaking at 800 rpm while adding 900 µL of ice-cold 70% ethanol drop-wise then incubated at 20 C for 2 h. Cells were further centrifuged for 5 min at − ◦ 8000 rpm, 4 ◦C, cell pellets were resuspended in 1 mL ice-cold PBS and the centrifugation was repeated then pellets were re-suspended in 1 mL of staining solution (PBS, 0.01% tween 80, 20 µg/mL PI, 1 µL/mL RNase [100 mg/mL]) and incubated at 37 ◦C for 30 min [43]. After washing and centrifugation, the cell suspension was vortexed and filtered using a polystyrene round bottom tube equipped with a cell strainer cap. The cell cycle distribution was then analysed using PE-A channel with a flow cytometer (BD Biosciences LSR II FACS, San Francisco, CA, USA) and 10,000 events per sample were acquired. The percentages of cells in the different cell cycle phases were analyzed in triplicate by the ModFit LT software (version 5.0).

3.8. Annexin V-FITC/PI for Apoptosis Detection Further analysis for apoptosis was performed using a FITC Annexin V Apoptosis Detection Kit according to the manufacturer’s instructions. Briefly, a total of 5.0 105 of HeLa cells were × cultured before being treated with the IC50 values, obtained from the MTT assay of the studied crude extracts, for 48 h. For the qualitative analysis, cells were seeded on the surface of a sterile glass coverslip placed in a twelve-well plate and incubated overnight before treatment. Treatment-free cells were grown as negative controls. Then, cells were washed twice with 1 binding buffer and further × incubated for 15 min with 200 µL of the binding buffer containing Annexin V-FITC and PI in the dark. Visualization of samples was carried out using a fluorescence microscope (CKX-53 Olympus, Tokyo, Japan). Apoptotic cells stained with green fluorescence and dead cells labelled with red fluorescence correlating to annexin V-FITC and propidium iodide, respectively. Four random fields of view for each sample were captured. For the quantitative analysis, the cells were harvested with trypsin and centrifuged at 300 g for × 10 min after treatment. Cells were then washed twice with 1 PBS buffer and further incubated with × 100 µL of the binding buffer containing annexin V-FITC and PI in the dark for 15 min. After that the samples were mixed with 400.0 µL binding buffer before being analyzed using a flow cytometer (BD Biosciences LSR II FACS). Results for three independent experiments expressed in a scatter plot as four different quadrants representing viable cells, necrosis, early and late apoptosis.

3.9. Caspase-Glo 3/7 Activity The influence of extracts on caspase 3/7 activity in HeLa cells was observed using Caspase-Glo 3/7 Assay kit (Promega). Following the manufacturer protocol, cells cultured in RPMI were seeded in 96-well plates overnight, then treated with the IC50 of the crude plant extracts obtained from the MTT assay and untreated cells as a control. After 24 h treatment, 100 µL of caspase reagent were added to each well, mixed and incubated for 1 h at room temperature. The luminescence was measured using a Fluostar Optima microplate reader (BMG Labtech, Ortenberg, Germany), then caspase activity was expressed as percentage of the untreated control within five replicates reading.

3.10. Analysis of Mitochondrial Membrane Potential MMP (∆Ψm) The mitochondrial membrane potential assay was performed by using tetramethylrhodamine ethyl ester (TMRE) to label active mitochondria. Simply, HeLa cells were seeded in 96-well plates (4 104) cells per well), after incubation overnight, cells were treated with IC of the four plant extracts × 50 obtained from the MTT assay and untreated cells worked as negative control. 24 h later, 50 µM of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was added to one sample replicate as Pharmaceuticals 2020, 13, 357 16 of 19 a positive control for 20 min, then cells were treated with 500 nM of TMRE and incubated for 20 min at 37 ◦C. Cells were washed twice with 0.2% bovine serum albumin (BSA) w/v in PBS, then fluorescence was detected using a Flurostar Optima microplate reader (λEx/λEm = 549/575 nm). For further evaluation of the disruption of MMP, visualization of the MMP analysis was done under a fluorescence microscope (CKX-53 Olympus). All experiments were performed in independent triplicates.

3.11. Wound Healing Assay Inhibition of cell migration and metastasis was evaluated by wound healing assay [44]. HeLa cells were seeded in 24 well plates. After 24 h, cells were treated with concentration values of IC50 of the different plant extracts obtained from the MTT assay and 0.1% DMSO (solvent control) for 2 h in serum-free medium. A scratch was made with a 200 µl pipette tip. Cells were then washed twice with ice-cold PBS (pH 7.4) and fresh medium was added. Wound closure was observed immediately (0 h) using an inverted microscope (CKX53, Olympus) and at different time intervals (24 h and 36 h). Cell migration and percentage wound healing were also calculated using Sketch and CalcTM® along with Gimp2.10.10® software measuring the distance between wound closures. The experiment was independently performed in triplicates.

3.12. Single Cell Gel Electrophoresis The assessment of the DNA damage and genotoxicity induced by the studied plant extracts was performed by the single-cell gel electrophoresis (Alkaline Comet Assay). All the procedures were performed in dark [45]. Briefly, 1 105 HeLa cells per well were seeded into a six-well plate and were × allowed to adhere overnight. Then cells were treated with IC50 of the different plant extracts obtained from the MTT assay and 0.1% DMSO (solvent control) for 24 h. Then the cells were trypsinized and centrifuged for 5 min at 1000 rpm to get the cell pellet. The obtained cell suspension was washed twice using sterile PBS (pH 7.4) and cell density was adjusted accordingly. After that, 80,000 cells (25 µL) of the treated and untreated cell suspension was mixed with 75 µL of 1% of pre-warm low melting agarose (LMA) (Carl Roth GmbH). The mixture was applied on the super frost glass slide previously pre-coated with 1% standard normal melting agarose (NMA) and was immediately covered with coverslips. The glass slides were then placed on an ice block for 10 min until solidified and the coverslips were gently removed. The cell membrane lysis was done by submerging the slides overnight into the staining jar containing cold lysis solution (300 mM NaOH, 1.2 M NaCl, 2% DMSO and 1% Triton X-100) [39]. The slides were then transferred to the electrophoresis tank containing alkaline electrophoresis buffer (300 mM NaOH and 1 mM EDTA) and were left in the buffer for 30 min to allow the unwinding of DNA. Electrophoresis was performed for 30 min at 250 mA current and 25 V, resulting in the DNA unwinding and exposing the alkali labile sites. After the electrophoresis, the slides were neutralized by washing the slides with double distilled water. The cell fixation was then done by submerging the slides into the 70% ethanol for 20 min. After fixation, cells were stained with SYBR® safe DNA staining dye (1:10,000 in PBS) for 20 min. Finally, the slides were washed with double distilled water to remove any unbound stains. The comet analysis was done under a fluorescence microscope (CKX-53 Olympus). Fifty individual comets were scored for each sample and analyzed using Comet Assay IV® software.

3.13. Statistical Analysis Non-linear curve fitting functions were applied on normalized dose-response cell viability data obtained from SRB and MTT assays, then IC50 values were calculated using GraphPad Prism 5. All the experiments were performed in triplicate unless otherwise stated and results are expressed as mean SD. One-way ANOVA was performed on data obtained of bar graphs using GraphPad Prism 5. ± Significance levels of p < 0.05 were considered for the rejection of the null hypothesis. Pharmaceuticals 2020, 13, 357 17 of 19

4. Conclusions In this study, the cytotoxic activity of extracts from four Cameroonian plants—XA, IC, EG and DP—was examined. These extracts could represent potential antitumor agents against the examined cancer cells in a concentration dependent manner. Cell cycle analysis showed the accumulation of hypodiploid cells in the sub-G0/G1 phase which is considered to be a marker for apoptotic cell death. As well, apoptosis was induced, by all of these extracts, with an increase in the caspase 3/7 activity and significant MMP disruption. These extracts introduce a promising option for inhibition of cancer cell metastasis. Further studies are warranted to identify the active constituents which are responsible for the anticancer properties and assessment of the dose-response relationship in vitro and in vivo.

Author Contributions: Conceptualization, A.S.; methodology, M.W. and J.P.D.; investigation, A.S., I.T., G.A., A.M.A. (Ahmed Mohamed Abdelsalam), and A.M.A. (Abdallah Mohamed Ayoub); data curation, A.S., and A.M.A. (Ahmed Mohamed Abdelsalam); software, validation and formal analysis, J.P.D., A.S., A.M.A. (Ahmed Mohamed Abdelsalam), and A.M.A. (Abdallah Mohamed Ayoub), writing-original draft preparation, A.S.; data curation and visualization, I.T., and A.S.; writing-review and editing, M.W., and U.B.; supervision and project administration, U.B. All authors have read and agreed to the published version of the manuscript. Funding: Authors would like to extend thanks to Yousef Jameel Foundation for providing the Scholarship funding and C. Mohr, Department of Pharmaceutics and Biopharmaceutics, Philipps University Marburg, for technical assistance in cell culture experiments. Acknowledgments: Authors would like to extend thanks to Yousef Jameel Foundation for providing the Scholarship funding and C. Mohr, Department of Pharmaceutics and Biopharmaceutics, Philipps University Marburg, for technical assistance in cell culture experiments. Conflicts of Interest: The authors declare no conflict of interest.

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