Aqueous Extract of Leaves and Flowers of Acmella caulirhiza Reduces the Proliferation of Cancer Cells by Underexpressing Some Genes and Activating Caspase-3

The increasing prevalence of cancers and the multiple side effects of cancer treatments have led researchers to constantly search for plants containing bioactive compounds with cell death properties. This work aimed at evaluating the antiproliferative effect of an Acmella caulirhiza extract. After evaluation of the in vitro antioxidant potential of the three extracts of Acmella caulirhiza (aqueous (AE-Ac), hydroethanolic (HEE-Ac), and ethanolic (EE-Ac)) through the scavenging of DPPH● and NO● radicals, the extract with the best antioxidant activity was selected for bioactive compound assessment and antiproliferative tests. Subsequently, the cytotoxic activity was evaluated on the viability of breast (MCF-7), brain (CT2A, SB-28, and GL-261), colon (MC-38), and skin (YUMM 1.7 and B16-F1) cancer lines using the MTT method. Then, the line where the extract was the most active was selected to evaluate the expression of certain genes involved in cancerogenesis by RT-PCR and the expression of cleaved caspase-3 involved in cell death mechanism by western blot. The AE-Ac showed the best scavenging activity with IC50s of 0.52 and 0.02 for DPPH● and NO●, respectively. This AE-Ac was found to contain alkaloids, flavonoids, and tannins and was more active on YUMM 1.7 cells (IC50 = 149.42 and 31.99 μg/mL for 24 and 48 h, respectively). Results also showed that AE-Ac downregulated the expression of inflammation (IL-1b (p = 0.017) and IL-6 (p = 0.028)), growth factors (PDGF (p = 0.039), IGF (p = 0.034), E2F1(p = 0.038), and E2F2(p = 0.016)), and antiapoptotic protein genes (Bcl-2 (p = 0.028) and Bcl-6 (p = 0.039)). The cleaved caspase-3 was positively modulated by the AE-Ac inducing thus cell death by apoptosis. AE-Ac showed inhibitory effects on the expression of genes involved in cancer progression making it a potential health intervention agent that can be exploited in cancer therapy protocols.


Introduction
Despite various technological advances, the cancer survival rate is still very low and is associated with about 9.96 million deaths worldwide [1].Breast cancer is the most common cancer with an incidence of 11.7%, followed by lung (11.4%), colorectal (10%), prostate (7.3%), stomach (5.64%), and other types of cancer (53.9%) [1].Te word "cancer" refers to a group of diseases that involve abnormal cell growth and invasion of adjacent or distant cells (or tissues).Various agents, both exogenous (radiation, viruses, or toxins) and endogenous (mutations), can afect the cell at several levels (genetic, biochemical, and epigenetic) and directly cause the deregulation of programmed cell death and initiate carcinogenesis [2].
Initiation of extrinsic apoptosis cell death pathway occurs when death receptors (Fas), tumour necrosis factor (TNF-α) receptors (TNFR1 and TNFR2), and TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 are occupied by their respective ligands [3,4].Te intracellular portions of death receptors possess a conserved protein-protein interaction domain known as the death domain, which is a binding site for adaptor proteins, such as the TNF receptor-associated death domain (TRADD) and the Fas-associated death domain (FADD), as well as initiator caspase 8 [5].Activated caspase-8 in turn stimulates efector caspase-7, enabling cleavage of the death agonist protein BH3, which translocates to the mitochondria and triggers cytochrome C release.In the cytosol, cytochrome C forms a multiprotein complex structure with apoptotic protease activating factor-1 (Apaf-1) and procaspase-9 so-called apoptosome.Te apoptosome permits the conversion of procaspase-9 to active caspase-9, which in turn contributes to the activation of efector caspase signaling, destroying the cell through apoptosis [5,6].In contrast to antiapoptotic proteins (Bcl-2, Bcl-6, etc.), proapoptotic proteins (Bcl-2 family) act by forming pores in the mitochondrial membrane to release cytochrome C [7].
Once cancer is initiated, the cells immediately begin to secrete several factors such as vascular endothelial growth factor (VEGF), transformative growth factor (TGF), metalloproteinase 2 and angiopoietin-1 (Ang-1) that promote the formation of the new vessels which permit cells to have nutrients, blood, and energy and thus escape chemotherapies [8].
Te anticancer treatments target several mechanisms through the use of antimetabolites (raltitrexed), alkylating agents (cyclophosphamide), topoisomerase inhibitors (doxorubicin), mitotic spindle poisons (vincristine), and cytotoxic agents (bortezomib), but all are accompanied by severe side efects (relapses, severe anaemia, and weight and hair losses).Researchers believed that the exploitation of the mechanisms of cell death remains a better alternative to cancer management.Tus, several medicinal plants such as Cola verticillata, Indonesian cucumber, and even those of the Amaryllidaceae family have been shown to have benefcial properties in the management of diseases including cancers [9][10][11].Tese plants exert their activities due to the presence of some bioactive compounds such as alkaloids and phenolic compounds.Alkaloids from the Amaryllidaceae family inhibit the independent efects of p53 on the proliferation of colon cancer cells [12].Phenolic compounds such as ampelopsin and apigenin through their antioxidant and anti-infammatory properties induce cell death by apoptosis, suppressing miR-512-3p and promoting the G1 phase of cell cycle involving the p27 Kip1 protein in glioma and breast cancer cells [13][14][15][16].Other bioactive compounds like tomentosin, a terpenoid isolated from plants of the Asteraceae family such as Inula viscosa, and jolkinolide B (extracted from Euphorbia kansui) inhibit the proliferation and migratory activity of cancer cells by downregulating the PI3K-Akt pathway and the expression of certain proinfammatory genes [17][18][19].Meilawati et al. [20] have shown that scopoletin, a coumarin present in most edible plants, exerts its anticancer activities through multiple mechanisms including the modulation of cell cycle arrest, the induction of apoptosis, and the regulation of multiple signaling pathways.Tus, Acmella caulirhiza, a fowering plant belonging to the Asteraceae family, is seasonally found in humid tropical areas.It is used as an ornamental plant but is also consumed as a vegetable by the people of Madagascar and Comoro Islands [21].Traditionally, the whole plant is used to fght respiratory diseases (cold, asthma, and tuberculosis), baby diaper rash, dental caries [22], and cancer [23].Studies also revealed that its crude extracts possess antiinfammatory, antimicrobial, and antioxidant properties [24] which are commonly noted in plants with proven anticancer potential [18,25], thus making Acmella caulirhiza a potential candidate in the treatment of cancer.Hence, the present study aimed at evaluating the antiproliferative properties of the aqueous extract of Acmella caulirhiza (AE-Ac) on some cancer cell lines.

Materials
2.1.1.Cell Lines.Te cancer cell lines used for this study as described in Table 1 were obtained from the cell bank of the Rothlin-Ghosh Lab, Howard Hughes Institute, Yale School of Medicine (USA).

Cell
Culture.Cells were grown in their respective media at 37 °C in a humidifed atmosphere with 5% CO 2 and 95% air.Te cells were trypsinized (0.1% trypsin) at 85% confuency.

Plant Material and Preparation of Extracts.
Te leaves and fowers of A. caulirhiza were collected in October 2018 at Bandjoun (West Region, Cameroon) and identifed at the National Herbarium of Cameroon (NHC) under the number 602 in comparison with specimen number 57420/ NHC of the herbarium.Te material was then sorted, cleaned, and dried until constant weight before being powdered and stored at room temperature in a tightly closed amber bottle.Te extracts were prepared according to the protocol of Fiardilla et al. [26] with slight modifcations.For the preparation of the aqueous, ethanolic, and hydroethanolic extracts, the same quantity of Acmella caulirhiza powder (100 g) was macerated in 1200 mL volume of distilled water (for 24 hours), 95% ethanol (for 72 hours), and a mix of water and 95% ethanol at a ratio of 1 : 1 (for 48 hours), respectively, at room temperature.Te supernatant of each mixture was collected by fltration using Whatman paper No. 3.Each resulting fltrate was frozen and then freeze-dried using a USIFROID SMH 45 (Lagep) to obtain the diferent extracts labelled AE-Ac for aqueous extract, HEE-Ac for hydroethanolic extract, and EE-Ac for ethanolic extract.
Te extract with the best antioxidant capacity was selected for further work.

Quantitative Phytochemical Analysis
(i) Estimation of total phenolic content: Te phenol content was evaluated using the method described by Singleton and Rossi [29].To 30 μL of extract (1 mg/ mL) prepared in an ethanol solution, 1 mL of Folin-Ciocalteu (0.2N) solution and 1 mL of sodium carbonate were added.Tirty (30) minutes after incubation at 25 °C, the absorbance was read at 750 nm.Gallic acid was used as standard and was treated in the same conditions as the extract.Te total phenolic content was expressed in microgram gallic acid equivalence/g of dry matter (μg GAE/g DM).(ii) Estimation of favonoid content: Te favonoid content was evaluated using the method described by Bohorun et al. [30].To one mL of the extract (1 mg/ mL), 1 mL of aluminum chloride (10%), 1 mL of potassium acetate (1 M), and 5.6 mL of distilled water were added.Te mixture was allowed to stand at 25 °C for 30 min.Te absorbance of the reaction mixture was read at 420 nm with a spectrophotometer.Catechin was used as the standard and treated in the same conditions.Te favonoid content was expressed in microgram catechin equivalence per gram of dry matter (μg CaE/g DM).(iii) Estimation of alkaloid content: Quantifcation of the total alkaloid content in the extract was performed according to the method described by Singh et al. [31] with slight modifcations.100 mg of extract was dissolved in 10 mL of ethanol solution (80%, v/v).Te mixture was homogenized and centrifuged for 10 min at 4000 g, 1 mL of the supernatant was introduced into a test tube, followed by the addition of 1 mL of acidifed FeCl 3 solution (FeCl 3 , 0.025 M; HCl, 0.5 M) and 1 mL of an ethanolic solution of 1,10-phenanthroline (0.05 M).Te mixture was then homogenized and incubated for 30 min at 100 °C in a water bath.Te absorbance of the reddish complex formed was read at 510 nm against the blank.Quinine (10 μg/mL) was used as standard, and the alkaloid content was expressed in microgram quinine equivalence per gram of dry matter (μg QiE/g DM).Biochemistry Research International (iv) Estimation of tannin content: Te method described by Bainbridge et al. [32] was used to estimate the total tannin content of the AE-Ac.In the protocol, 1 mL of the extract (1 mg/mL) was mixed with 5 mL of working solution (50 g of vanillin +4 mL of HCl in 100 mL of distilled water) and the mixture was incubated at 30 °C for 20 min.Te absorbance was read at 500 nm against the blank.Gallic acid (0-1000 μg/mL) was used as standard, and the calibration curve was used to compute the tannin content of the extract.Te results were expressed in micrograms of gallic acid equivalence per gram of dry matter (µg GAE/g DM).(v) Estimation of total terpenoid content: Total terpenoids were determined according to the method of Ghorai et al. [33].To 500 mg of extract was added 3.5 ml of ice-cold 95% methanol.It was homogenized before centrifugation at 4000 g for 15 min at room temperature and the supernatant was collected.To 200 μl of supernatant, 1.5 ml of chloroform was added, and the mixture was then roughly mixed and left to stand for 3 minutes.Ten, 100 µl of sulfuric acid was added and the whole mixture was incubated at room temperature for 2 h in the dark.Ten, carefully and gently the supernatant was decanted without disturbing the precipitation.Ten, 1.5 ml of 95% methanol was added and vortexed until the precipitation completely dissolved in the methanol and read at 538 nm.Results were expressed using linalool as a reference molecule.

Cell Viability Assay.
Cell viability was performed using the thiazolyl blue tetrazolium bromide (MTT) according to Ahmad et al. [34] with some modifcations.About 5000 cells of B16F and YUMM 1.7; 10000 of CT2A, MC-38, and SB-28; 15000 of GL231; and 20000 MCF-7 were plated per well in 96-well plates overnight.Tey were then treated with the best antioxidant capacity extract at 5, 10, 50, 100, 250, or 500 μg/mL and incubated for 24 h or 48 h.MCF-7 cells were also treated for 72 h (based on the fact that its half-life was about 42 hours).At the end of the diferent incubation periods, the treated media were removed and 10 μL of 5 mg/ mL MTT solution was added to each well and then incubated for 4 h.Later, 100 μL of DMSO was added to each well and plates were protected from light for incubation overnight.

Antioxidant Potential of Extracts of Acmella caulirhiza.
Te NO • and DPPH • radical scavenging potential of the diferent extracts of A. caulirhiza is reported in Figures 1(a) and 1(b).Results showed that the extracts scavenged these radicals in a concentration-dependent manner.Te AE-Ac presented the highest scavenging capacity towards NO • and DPPH • with SC 50 of 0.02 and 0.52, respectively, followed by the HEE-Ac (Table 3).As the tumour microenvironment is characterized by a high oxidative stress state, the AE-Ac with the best antioxidant activity was selected for further study.

Bioactive Content of the Aqueous Extract of Acmella caulirhiza.
Results revealed the presence of total phenolics, alkaloids, and favonoids in the aqueous extract of A. caulirhiza (Table 4).

Efect of AE-Ac on Cell Viability.
A concentrationdependent efect of the AE-Ac was noted on the proliferation of some cell lines as reported in Figures 2(a)-2(g).Te CT2A, SB-28, MC-38, and MCF-7 cell lines were the most resistant to the AE-Ac as their IC 50 s were higher than 500 μg/mL after 24 h of exposition.However, after 48 h of treatment, their IC 50 s were >500, 351.5).As the antiproliferative properties of the extract were more perceptible on the YUMM 1.7 cell line, it was selected for the evaluation of the extract ability to regulate the expression of certain genes.

Efect of AE-Ac on the Lipid Peroxidation.
Te RSL3 induced peroxidation in YUMM 1.7 cells, but the extract signifcantly limited the peroxidative properties of RSL3 for all three concentrations.Nonetheless, this protective property was lower than that of ferrostatin-1 (p < 0.05) (Figure 3).Biochemistry Research International

Efect of the AE-Ac on the Expression of Some Genes
Involved in Carcinogenesis of the YUMM 1.7 Cells

Efect of the AE-Ac on Proinfammatory Gene
Expression.Except for IL-10, treatment of YUMM 1.7 cells with the aqueous extract of A. caulirhiza led to a decrease in the expression of TNF-α, IL-1b (p � 0.017), and IL-6 (p � 0.028) genes compared to control (Figure 4).

Efect of AE-Ac on Two Genes of Antiapoptotic Proteins.
After exposure of YUMM cells to AE-Ac, a signifcant downregulation of Bcl-2 (p � 0.028) and Bcl-6 (p � 0.039) gene expression was observed (Figure 6).

Efect of AE-Ac on the Cleaved Caspase-3's Expression.
Results revealed that the AE-Ac induced apoptosis just like etoposide (an inducer of apoptosis) in cells through the activation of cleaved caspase-3, a cysteine protease involved in cell death by nucleic acid degradation (Figure 7).

Discussion
Cancer cells are unable to control the expression of cell death genes, making them resistant to chemotherapies.Anticancer therapies target several mechanisms of cell death through the use of antimetabolites, alkylating agents, mitotic spindle poisons, and even cytotoxic agents.However, these therapies are toxic to cancer cells and also to normal cells of the body.Te control of natural mechanisms of cell death remains a better alternative in the management of cancers.Results of this study which aimed at evaluating the antiproliferative properties of the aqueous extract of Acmella caulirhiza revealed that the extract exerted antiproliferative properties through the downregulation of the expression of some genes involved in the cancerogenesis pathway and this was due to the presence of bioactive molecules.Te evaluation of the in vitro antioxidant potential (scavenging of DPPH • and NO • radicals) of the three extracts of Acmella caulirhiza (EE-Ac, HEE-Ac, and AE-Ac) showed that the AE-Ac exhibited the best free radical    8 Biochemistry Research International scavenging activity (Table 3).Tis antioxidant capacity of the extract is linked to its high content of phenolic compounds as well as other bioactive compounds like alkaloids (Table 4).Phenolic compounds have free hydroxyl groups and conjugated double bonds in their structures capable of providing hydrogen or electron to a free radical or a metal [35].Also, it is well known that the bioactive compounds of plants have already demonstrated anticancerous properties in both in vitro and in vivo studies [11,36,37], thus the selection of the aqueous extract for antiproliferative tests.One of the biological characteristics of cancer cells is the production of reactive oxygen species.One of the major drawbacks of chemotherapy is lipid peroxidation, which is frequently caused by interactions of these species with polyunsaturated fatty acids in lipid membranes.Lipid peroxidation is considered a key biochemical process in the toxicity process that causes cell death as well as oxidative damage to cellular components.During this process, free radicals steal electrons from cell membrane lipids, which jeopardize cell life by causing decreased membrane fuidity, increased membrane permeability, and decreased physiological performance [38].In this study, the potential of AE-Ac to inhibit lipid peroxidation was measured by evaluating the viability of YUMM 1.7 line cancer cells exposed to RSL3 (a lipid peroxidation activator), which acts by inhibiting glutathione peroxidase 4. AE-Ac at all concentrations (50, 100, and 150 μg/mL) showed protective efects of cell membranes against RSL3 by protecting cells from the peroxidative action of RSL3.Tis activity could be explained by the antioxidant properties of the aqueous extract of Ac through its ability to scavenge free radicals generated by tumour cells, thus preventing the peroxidation of lipid membrane [38].
Te evaluation of the cytotoxic properties of this aqueous extract on breast, brain, skin, and colon cancer cell lines showed that the extract was more active on YUMM 1.7 cells with IC 50 s of 149.42 and 31.99 after 24 and 48 h, respectively (Table 5).Xie et al. demonstrated that alkaloids, favonoids, coumarins, and terpenoids also present in our extract upregulated the expression of Kip/p27 leading to a decrease in cyclin-D, cyclin-E, and CDK2/4/6 proteins in the melanoma cancer cell lines (WM1361B and WM983A) [16], colon cancer cell lines (HCT-116, LoVo, and DLD-1) [12], and human breast cancer cell line (MCF-7) [37].Tis could lead to the breakage of retinoblastoma and E2F proteins, stopping the cell cycle in the G1/G0 phase.
Tis downregulated the transcription factors NF-κB and upregulated tumour suppressor factors (p53 and p21t) which are cellular gatekeepers of growth.
Cleaved caspase-3 is well known as an executor protease of cell death by apoptosis through the reduction of mitochondrial membrane potential.Te AE-Ac induced expression of this protein (Figure 7).Te favonoids in the extract could reduce mitochondrial membrane potential, leading to the release of apoptogenic factors such as Arts, Diablo, Second Mitochondria-Derived Activator of Caspase (SMAC), and High-Temperature Requirement Protein A2 (Omi/HTRA2).Tese proteins can block the action of the apoptosis inhibitor proteins Bcl-2 and Bcl-xL (which inhibit apoptosis by forming macropores on the mitochondrial membrane and prevent the release of cyt c and the formation of the apoptosome) and the activation of caspase-3 [39,40].Once activated, caspase-3 translocates to the nucleus in its cleaved caspase-3 form where it causes cell death by DNA fragmentation and degradation of nucleic acids [6,41].Indeed, Bernard et al. have shown that cleaved caspase induced gene transcription by interacting with their promoters and inhibiting their expression within the VEGFA gene in the chip experiment [42,43].Tis resulted in the downregulation of several pathways of angiogenesis (FAS, TRAIL, IFN-c, TNF receptor, and RAC1) in MCF-7 and human Jurkat leukaemia cells [40,44].In addition, studies had shown that apigenin (a favonoid) exerted antiproliferative activities on human melanoma cell lines A375P and A375SM through the activation of the apoptotic pathway and the decrease of the antiapoptotic protein Bcl-2 expression [15,45].
To escape cell death, cancer cells also alter the expression of genes and proteins associated to infammation.Some cytokines and proinfammatory proteins like TNF-α and IL are promoters of carcinogenesis through the activation of several signaling pathways [3,7,46].PCR analyses showed that treatment of YUMM 1.7 cells with the AE-Ac downregulated the expression of the proinfammatory gene (TNF-α, IL-1β, and IL-6) (Figure 4).Mathieu et al. demonstrated that alkaloids such as those of Amaryllidaceae (lycorine, narciclasine, and haemanthamine) exert anticancer activities through the inhibition of NFkB (an oncogene involved in tumorigenesis and resistance to apoptosis [47]) [12].It consequently activated the p53 tumour suppressor gene leading to a decrease in the expression of TNF-α, IL-6, IL-1β, and VEGF in colorectal cancer cells [12].On the other hand, growth factors (IGF-1, TGFβ, VEGF...) are activated by binding to their respective intracellular receptors, which can activate several signaling pathways involved in cell proliferation [8].PCR results showed that growth factors' gene expression was downregulated after 4 h of treatment (Figure 5).Tis would be due to the presence of favonoids which can prevent the binding of ligands to their membrane receptor such as epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK), thus inhibiting their activity with the consequence of stopping the cell proliferation [48,49].
Te deregulation of B cell lymphoma family proteins is the main feature of malignant diseases.It is responsible for resistance to cell death and thus to treatment [7,50].In this study, exposure of cells to EA-Ac resulted in a downregulation of the expression of antiapoptotic genes Bcl-2 and Bcl-6 (Figure 6) due to the capacity of the terpenoids present in the AE-Ac.Indeed, Yang et al. had shown that borneol, a bicyclic monoterpenoid, caused a signifcant release of cyt c which promotes apoptosome formation by aggregating caspase-9 with Apaf-1 in the cytosol triggering apoptosis [16,18].Results of western blot analysis (Figure 7) show that the extract also induced Biochemistry Research International death by apoptosis through its ability to promote activation of caspase-3.

Conclusion
Te AE-Ac exhibited the highest antiradical scavenging activities due to the presence of bioactive compounds (alkaloids, favonoids, tannins, and terpenoids).Tis extract was more cytotoxic on YUMM 1.7 cells after 24 and 48 h incubation periods compared to the other cancer cell lines.Te AE-Ac can induce cell death through the underexpression of infammation, growth factors, and antiapoptotic protein genes.Te presence of cleaved caspase-3 after treatment of YUMM 1.7 cells with the extract confrmed its capacity to induce apoptosis.Studies of other antiproliferative pathways of this extract can reinforce the knowledge of the aqueous extract of Acmella caulirhiza as a potential candidate for cancer treatment [51].

Figure 6 :Figure 7 :
Figure 6: Efect of AE-Ac on some genes of antiapoptotic proteins in YUMM 1.7 cells.Bcl: B cell lymphoma.* Signifcant diference at p < 0.05 compared to the control.

Table 1 :
Cell lines and description.
Te absorbance was read at 570 nm.Signifcance was set at p < 0.05.Te IC 50 and SC 50 values were obtained by linear regression and Microsoft Excel 2016 spreadsheet software was used to plot the graphs.
2.5.Efect of the Extract on the Expression of Some GenesInvolved in Cancerogenesis.Te YUMM 1.7 cells (350000/ well in a 6-well plate) were treated with 150 μg/mL of AE-Ac in triplicate and incubated for 24 h.RNA extraction and purifcation were carried out for each treatment using the RNeasy Mini Kit (QIAGEN, USA).RNA quantifcation was done using the NANODROP 2000 Spectrophotometer of Termo Fisher Scientifc.Te purifed RNA was then used to synthesize cDNA using Bio-Rad's iScript cDNA Synthesis Kit.For qPCR analyses, the KAPA SYBR FAST Universal kit with corresponding primers was prepared for each gene in triplicate.Te C1000 Touch Termal Cycler of Bio-Rad with associated software was used to run qPCRs.All kits were used according to manufacturers' protocols.Te genes evaluated included growth factor (PDFG, IGFR-1-R, TGFβ, VEGF, E2F1, and E2F2), antiapoptotic (Bcl-2 and Bcl-6), and cytokine (TNF-α, IL-1b, IL-6, and IL-10) genes.2.6.Efect of the Extract on the Expression of Cleaved Caspase-3.Following a 24 h of treatment of YUMM 1.7 cells (350000 cells per well in a 6-well plate) with 150 μg/mL of extract or 2 μM of etoposide, cells were lysed using RIPA bufer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 2 mM EDTA).Protein content was determined using the BCA assay kit (Termo Fisher Scientifc) according to the manufacturer's instructions.Samples (nontreated cells and cells treated with either extract or etoposide) were loaded and run in a polyacrylamide gel for 90 min, at 100 V. Separated proteins were then transferred to a PVDF membrane and blocked using Intercept Blocking Bufer (LI-Biochemistry Research International COR).Te membrane was then incubated with a primary antibody from rabbit (caspase-3, Cell Signaling Technology) and mouse antibody for β-actin (Cell Signaling Technology) for a period of 24 h in a cold room.Te membrane was then washed 3 times with PBST (PBS + 0.1% Tween 20) and incubated with the secondary antibody; the donkey antirabbit antibody (cell signalling technology) was conjugated to a chemiluminescent such as IR680 (red) to detect cleaved caspase-3, and the donkey antimouse antibody conjugated to IR800 (green) to reveal β-actin.Te plate protected from light was incubated for 1 h and later visualised using the Odyssey system associated with ImageStudio (LI-COR).2.7.Statistical Analysis.Data were expressed as mean-± standard deviation (SD).Statistical analysis was performed using Statistical Package for Social Science (SPSS) software version 20.0 for Windows.One-way analysis of variance (ANOVA) and the least signifcant diference (LSD) post hoc test were used to compare means between groups.

Table 2 :
Primers of the genes used (Sigma-Aldrich DNA oligos template).

Table 4 :
Content of bioactive compounds in the aqueous extract of Acmella caulirhiza.

Table 5 :
Inhibitory concentrations 50 of the AE-Ac on cancer cell lines.