THEREOF

Technical Field

The invention relates to use of ferruginin A (FGA) compound that presents an effective cytotoxic activity in cancer chemotherapy as an anticancer agent and MAPK pathway inhibitor due to biological activity thereof.

Prior Art

Ferruginin A naturally occurring anthranoid derived through isolation from the bark of Harungana madagascariensis. It is known from various publications available in the literature that FGA is a compound presenting cytotoxic effect.

In the state of the art, Sorafenib compound (C21H16CIF3N4O3) is a kinase inhibitor and is used prevalently for treatment of the primary kidney cancer, terminal primary liver cancer and radioactive iodine resistant terminal thyroid carcinoma. Although the compound produces good results in treatment of cancer, majority of the patients develop resistance against sorafenib after some time, which increases the requirement for new MAPK inhibitors for cancer chemotherapy.

The study performed by Breiner-Goldstein et al. known in the state of art discloses targeting of anthracycline-resistant tumor cells with synthetic aloe-emodine glycosides. A natural antronoid that easily infiltrates the anthracycline-resistant tumor cells, the cytotoxic activity of aloe-emodine (AE) is developed with an amino-sugar unit attached to the anthraquinone core thereof. The new class of AE glycosides (AEGs) present a significant advancement over up to more than 2 orders of magnitude greater than AE’s and clinically used anthracy cline doxorubicin (DOX) against many cancer cell lines with distinct DOX resistance levels in the cytotoxic sense.

The study performed by Alvarez et al. known in the state of art studies the antioxidant activity of the phenolic content extracted from the fruits of two species of Vismia Genus (GUTTIFERAE) plant. The vegetal material derived from the dried and pulverized fruits of Vismia baccifera ssp ferrugine and V. Guianensis plants has been extracted through successful filtration with petroleum ether, ethyl acetate and methanol. Two antronoids (ferruginin A and g-hydroxyferruginin A) and one anthraquinone (vismiaquinone) in the extract were isolated and their structures have been identified through spectroscopic methods and by comparison with the values available in the literature.

The study performed by Nascimbeni et al. known in the state of art mentions of an investigation conducted on intestinal obstruction, antronoid laxatives (intestinal obstruction medication), Melanosis Coli and colon cancer. The relationship between the colorectal cancer (CRC) and intestinal obstruction, use of antranoid laxatives and melanosis coli is currently under debate. Abnormal crypt foci (ACF) are the microscopic lesions of the colonic mucosa with suspicion of preneoplastic formation, and it is argued that the research on these lesions determines the cause and effect relation between presumptive risk factors and CRC. Therefore, this study investigates use of the relationship between sigmoid cancer (SC) and intestinal obstruction, use of antranoid laxatives and melanosis coli in ACF analysis. The conducted study verifies the relation between colon cancer and ACF density and not endorses the hypothesis that there is cause and effect relation between CRC and the intestinal obstruction, use of antranoid laxatives and melanosis coli.

The United States patent document no. US5227385, one of the publications known in the state of art, mentions of a method for treating neurodegenerative diseases. The invention makes use of a ferruginine ingredient and an anhydrous-ingredient at effective rates at patients suffering from neurodegenerative diseases. Said ingredients can function as racemic mixture or as enanti omonomer s .

The Turkish patent application no. TR 2011/00961 (Turkish registration of the documented European patent application no. EP1446381B1), one of the publications known in the state of art, mentions of anthranilic acid amides and the pharmaceutical use thereof. The invention relates to a novel anthranilic acid amide derivate, the processes for preparation thereof, application thereof in a procedure for treatment of human and animal body, usage thereof either alone or in combination with one or more active substances for treatment of a neoplastic disease, particularly of a tumor disease, retinopathies and age related macular degeneration and usage thereof either alone or in combination with one or more pharmaceutically active substances for production of a pharmaceutical preparation for treatment of a neoplastic disease, retinopathies and age related macular degeneration.

Brief Description of the Invention

The objective of the invention is to perform identification of the biological activity of Ferruginin A (FGA) compound, a natural anthranoid that can be isolated from the bark of Harungana madagascariensis, as a chemotherapy agent and MAPK pathway inhibitor.

Another objective of the invention is to take advantage of the fact that Ferruginin A presents low effect on human normal skin fibroblasts, thus presents selective toxicity towards the cancer cells selective toxicity.

Another objective of the invention is to take advantage of the fact that Ferruginin A induces apoptosis in MCF-7 cell line and that it immobilizes the cell cycle in Go/Gl phase.

Another objective of the invention is to take advantage of the fact that Ferruginin A induces MMP modification at MCF-7 cell line.

Another objective of the invention is to take advantage of the fact that Ferruginin A induces ROS production at MCF-7 cell line in significant levels.

Detailed Description of the Invention

“Use of Ferruginin A (FGA) compound as a chemotherapy agent and MAPK pathway inhibitor due to biological activity thereof’ performed for achieving the objective of the present invention is illustrated in the enclosed figures; wherein:

Fig. 1 - Illustrates the view of the flow diagram of Ferruginin A isolation process.

Fig. 2 - Illustrates the MAPK pathway genes the expression of which is modified the most upon application of Ferruginin A.

Fig. 3 - Illustrates the view of the cell cycle distribution in the Ferruginin A applied

MCF-7 cells (cell cycle distribution of the control MCF-7 cells (control); cell cycle distribution of MCF-7 cells to which the 0.19 pg/mL (la), 0.10 pg/mL (lb) and 0.05 pg/mL (lc) ferruginin A is applied for 72 hours.) Fig. 4 - Is the graphical illustration of the impact of Ferruginin A to the mitochondrial membrane potential at MCF-7 cells ((a) Control; (b) Ferruginin A (FGA): 0.19 pg/mL ferruginin A at 72 hours)

Fig. 5 - Is the graphical illustration of the activity of caspase 3/7 and caspase 9 enzymes achieved as a result of applying Ferruginin A to MCF-7 cells for 6 hours.

Fig. 6 - Is the graphical illustration of the induction of reactive oxygen species level due to application of ferruginin A to the MCF-7 cells (Ferruginin A has been applied at an amount of 0.19 pg/mL and 0.10 pg/mL corresponding to ICso and ½ IC5 ₀ values for 24 hours).

The denomination of the parts in the figures has been abbreviated as set forth hereunder;

HM-BP. Harungana madagascariensis- Bark Powder

MeOH-E. Methanol Extraction

HMB. Bark Extract

HMBa. Bark Extract-Hexane fraction

HMBb. Bark Extract-Dichloromethane fraction

HMBc. Bark Extract-Fraction from Methanol

HMBal. Bark Extract-combination of 1-3 sub fractions of Hexane fraction

HMBa2. Bark Extract-combination of 5-15 sub fractions of Hexane fraction

HMBa3. Bark Extract-combination of 16-79 sub fractions of Hexane fraction

HMBa4. Bark Extract-combination of 80-95 sub fractions of Hexane fraction

HMBa5. Bark Extract-combination of 96-132 sub fractions of Hexane fraction

The present invention is to use Ferruginin A (FGA) compound, a natural anthranoid and a cytotoxic substance that can be isolated from the bark of Harungana madagascariensis, for the purpose of exerting biological activity as a chemotherapy agent and mitogen-activated protein kinase (MAPK) pathway inhibitor for the purpose of impelling the cells to necrosis through apoptosis through immobilizing the cell cycle of the cancer cells depending on the toxicity thereof.

Tinder the scope of the invention, Ferruginin A (FGA) compound is used at the concentration in the range of 0,1-3 pg/ml in order to impel the cells to necrosis through apoptosis by immobilizing the cell cycle of the cancer cells at colon cancer, lung cancer and liver cancer and for inhibiting the MAPK pathway.

Method for Identification of the biological activity of Ferruginin A (FGA) Compound

A method for identification of the biological activity of Ferruginin A (FGA) compound, A natural anthranoid and cytotoxic substance, as a chemotherapy agent and mitogen-activated protein kinase (MAPK) pathway inhibitor for the purpose of impelling the cells to necrosis through apoptosis through immobilizing the cell cycle of the cancer cells depending on the toxicity thereof, wherein the method comprises the process steps of:

- Isolating FGA from the bark of Harungana madagascariensis Lam. ex Poir.

(Hypericaceae),

o Extracting the dried plant barks with methanol (the amount of processed plant barks and leaves is 1-5 kg. and 1-5 L methanol was used for extraction from the barks and 0.1-1 L methanol was used for extraction from the leaves).

o Concentration thereof under vacuum at low pressure in order to produce black residue from methanol extracts (from the leaves (11.25% by weight) and the bark 8.67%)),

o Obtaining the filtrates by drying with hexane and dichloromethane, respectively, after dissolving 130 g bark extract (HMB) in 1 L methanol, o Concentrating the filtrates with low-pressure vacuum, thus separating into two fractions as the Bark Extract-Hexane fraction (HMBa) (HMBa derived from 15 g hexane) and the Bark Extract-Dichloromethane fraction (from 25 g dichloromethane),

o Grouping the sub-fractions obtained as a result of silica gel chromatography of the bark Extract-Hexane fraction as Bark Extract- combination of 1-3 sub fractions (HMBal), Bark Extract-combination of 5-15 sub fractions of Hexane fraction (HMBa2), Bark Extract-combination of 16-79 sub fractions of Hexane fraction (HMBa3), Bark Extract-combination of 80-95 sub fractions of Hexane fraction (HMBa4) and Bark Extract-combination of 96-132 sub fractions of Hexane fraction (HMBa5),

o Purification of the Bark Extract-combination of 16-79 sub fractions of Hexane fraction (HMBa3) through silica gel column chromatography using graduated mixture of hexane-acetone, and o Isolation of FGA through such purification (in particular, fractions no. 14-15 are used in FGA isolation as such fractions contain 15 mg FGA)

- Identification of the effect of the isolated FGA to the expression of MAPK genes at breast cancer cells (MCF-7 cells) through relative quantification method,

- Identification of the in vitro cytotoxic activity of FGA through neutral red uptake test,

- Identification of the cancer cells where the FGA cytotoxicity is intrinsic and the efficacy thereon through the neutral red uptake test, and

Identification of the FGA’s effect on the cell cycle and on the DNA content in MCF-7 cells, identification of the caspase 3/7 and caspase 9 activities through caspase activity tests, and identification of the FGA’s effect on mitochondrial membrane potential (MMP), and on the generation of reactive oxygen species (ROS).

Identification of the effect of the isolated FGA to the expression of MAPK genes at breast cancer cells (MCF-7 cells) is performed as set forth hereunder:

1. The total RNA has been isolated from the cells in the control and test group,

2. cDNA synthesis has been performed

3. SYBR Green based qPCR has been performed, and

4. The compiled data has been assessed using relative quantification and the expression of the MAPK pathway genes has been compared.

Neutral red intake test has been carried out for determining the cytotoxic effect of the FGA. In brief, the cells were cultivated in 96-well plate and the quantity of FGA that reduces to half the viability of the cell to which certain doses of FGA had been applied has been determined.

In order to determine the impact of the FGA on the expression of MAPK genes (Gene expression; synthesis of mRNA molecule coded by the gene through transcription of said gene) in MCF-7 cells, the RT ² Profiler™ PCR Array Human MAP Kinase Signaling Pathway (PAHS-061A) was used based on the protocol provided by the producer of said kit (Qiagen, Hilden, Germany) and the variations are observed. As a result of the observations, it is seen that the gene that increases the most is CDKN2B by 2.52 times, while the genes that reduce the most are CDK4, CDKN1B, CREB1, KRAS, TP53 (8-fold), ATF2 and B2M (l0.88-fold), CCNA2, CCNB1 (l2.7-fold) and ACTB (25.4-fold).

Neutral red intake test has been carried out for determining the in vitro cytotoxic activity of the FGA. It is observed that the FGA produces ICso values at all tested cancer cell lines at values equal to or less than the amount of 4 pg/mL. ICso represents the drug concentration that causes the cell quantity to reduce by half. Under the scope of the invention, the level of reactive oxygen species (ROS) at ICso concentration and the level of ROS at ½ ICso, that is to say half-IC ₅ o concentration has been determined and presented in graphical form (Figure 6). The process for determining the ROS production has been carried out as outlined hereunder: the cells were cultivated in 96-well plate and the reactive oxygen species so produced, to which FGA is applied at certain rates, were measured fluorometrically. During the empirical studies, the ICso values of the FGA in SPC212 (0.20 pg/mL) cells, i.e. lung carcinoma cells, and in MCF-7 (0.19 pg/mL) cells, i.e. breast cancer cells, is below 1 pg/mL. It was observed that the FGA presented selective toxicity through more than 3 times the selectivity index by presenting low effect on normal fibroblast CRL2120 cells.

The effect of FGA on the cell cycle and DNA content at MCF-7 cells after application for 72 hours has been analyzed using BD FACVerse™ System at the flow cytometry based on the directives provided by the producer of BD Cyclotest™ Plus DNA Reagent kit. It is observed that FGA triggers apoptosis depending on the dose and that it immobilizes the cell cycle at GO/G1 phase. The apoptotic cell ratio, which is 3.1% in the control group, reaches up to 19.8%; 16.4% and 12.8% after application of FGA for 72 hours at concentrations of 0.19 pg/mL (IC50), 0.10 pg/mL (½ x IC50) and 0.05 pg/mL (¼ x IC50), respectively.

In determination of the effect of FGA application in MCF-7 cells for 72 hours on MMP, the analysis is performed using flow cytometry according to the instructions for use of 5, 5', 6,6'- tetrachloro-l,l',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-l; Biomol, Germany) staining test kit (BD™ MitoScreen Mitochondrial Membrane Potential Kit). When compared to the control group, it is observed that application of FGA induces loss of MMP at MCF-7 cells, and that the loss of MMP, which is 4.3% at the control group, reaches up to 15.8% after application of FGA for 72 hours at a concentration of 0.19 pg/mL (IC50). This indicates that apoptosis triggered at MCF-7 cells with the impact of FGA occurs due to loss of MMP.

In observation of the caspase 3/7 and caspase 9 activities of FGA at MCF-7 cells, the Caspase-Glo Assay kit (Promega, Mannheim, Germany) is used according to kit instructions and it is observed that FGA did not trigger caspase 3/7 and caspase 9 activities during apoptosis. In order to determine the impact of FGA on ROS production in MCF-7 cells, fluorescence analysis has been performed in Molecular Device SpectraMax M2 Absorbance/Fluorescence Microplate Reader through 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma-Aldrich, Germany) staining using the protocol provided by the manufacturer. It is seen that FGA allows an increase of ROS by more than 4 times (at IC50 value) in MCF-7 cells, thus triggering a significant level of ROS production. This indicates that apoptosis triggered at MCF-7 cells with the impact of FGA occurs due to activation of ROS production.

The Ferruginin A (FGA) compound, the effectiveness of which is identified under the scope of the invention, can be administered to the patient in the form of tablet or capsule, as well as in the form of oral drops.

Ferruginin A (FGA) compound is a cytotoxic substance, wherein such toxicity is peculiar to the cancer cells, and such toxicity immobilizes/pauses the cell cycle of the cancer cells and impels the cells to necrosis through apoptosis. Ferruginin A (FGA) compound is a MAPK pathway inhibitor. The neutral red intake test demonstrated that FGA presents higher toxic effect on the cancer cells on which its cytotoxicity is intrinsic (that it has less toxic effect on the normal cells when compared to the cancer cells it is effective on). In brief, the cells were cultivated in 96-well plate and the quantity of FGA that reduces to half the viability of the cell to which certain doses of FGA had been applied has been determined.

Under the scope of the invention, use of Ferruginin A (FGA) as a MAPK pathway inhibitor is predicates on the fact that it is used as a cytotoxic compound that lyses the cancer cells as it is less toxic to the normal cells compared to the cancer cells. Such characteristics indicate that Ferruginin A can be a chemotherapy/anticancer agent with significant potential. Ferruginin A is a natural anthranoid that can be isolated from the bark of Harungana madagascariensis, and its effectiveness as MAPK pathway inhibitor in MCF-7 cells forms basis for development of the present invention. Ferruginin A is a potential cytotoxic compound, which can be used for chemotherapy at colon, lung, breast and liver cancers. Ferruginin A has low impact on human normal skin fibroblasts, thus presents selective toxicity towards cancer cells. Therefore, the compound is very suitable for use as an anticancer agent. Ferruginin A induces apoptosis at MCF-7 cell line and immobilizes the cell cycle at Go/Gl phase, causes MMP modification at MCF-7 cell line and induces production of ROS at significant levels at this cell line. The method of identification of the biological activation based on use of Ferruginin A under the scope of the invention has been developed as an anticancer agent from the class of MAPK inhibitors, such as sorafenib, available in conventional applications. It is observed that the FGA addressed under the scope of the invention is a MAPK pathway inhibitor, and its toxicity against the normal cell is far too less when compared to the toxicity against the cancer cells, and accordingly, it is proposed as a solution method against resistance to sorafenib, which develop at the patients in the conventional treatments.

The fact that the number of MAPK inhibitors that can be used for chemotherapy in the state of art is extremely scarce makes use of FGA for chemotherapy extremely important. The fact that FGA is effective in a broad spectrum allows it to feature good selectivity index. Furthermore, as isolation of FGA is relatively easy process, and the low cost of the medication to be produced and at larger quantities compared to the anti-cancer formulations used today reveals its ability to respond to broader needs in economic terms.

Empirical Studies

Isolation of Ferruginin A (FGA) is schematically illustrated in Figure 1. FGA is isolated from the bark of Harungana madagascariensis Lam. ex Poir. (Hypericaceae). Harungana madagascariensis was picked from Dschang, west of Cameroon (5°27'N 10°04Έ) in February 2012 under the scope of the empirical studies carried out for development of the invention. The plant picked was submitted to National Herbarium (Yaounde, Cameroon) and registered under registry number of 43848/HNC. The dried plant barks (1.5 kg) (200 ml (leaves) and 5 L (bark)) was extracted using methanol. The methanol the barks, and 0,1-1 L methanol was used for extraction from the leaves), The methanol extracts (from the leaves (11.25% by weight) and the bark 8.67%)) were concentrated under vacuum at low pressure in order to produce black residue. Upon applying low-pressure vacuum, the methanol extracts produced black residue at the concentration of 11.25% by weight from the leaves, and at the concentration of 8.67% by weight from the barks. After dissolving the bark extract (HMB) (130 g) in methanol, the extract was then dissolved in hexane and dichloromethane, respectively. The filtrates are then concentrated under low-pressure vacuum and subdivided into two fractions as Bark Extract-Hexane fraction (HMBa) (from 15 g hexane) and Bark Extract-Dichloromethane fraction (HMBb) (from 25 g dichloromethane). As it is discovered that the fraction remaining from the Bark Extract-Methanol (HMBc) features low antibacterial activity, this fraction is excluded from further study. Then, silica gel column chromatography is applied to Bark Extract-Hexane fraction (HMBa) and 132 sub-fractions with elution solution concentrations and volumes of Hex (100%; 2750 mL), Hex-CH ₂ Cl2 (95:5; 1500 mL), Hex-CHiCk (90: 10; 1000 mL), Hex-CTECk (85: 15; 1000 mL), Hex-CTECk (75:25; 9000 mL), Hex-QECk (65:45; 3000 mL), Hex-QECk (50:50; 6000 mL), Hex-QECk (40:60; 1250 mL), Hex-QECk (30:70; 1000 mL), CH2CI2 (100%; 250 mL), CTECk-chloroform (CH3OH) (95:5; 1750 mL), and CftOH-ethyl acetate (EtOAc) (85:15; 4500 mL), respectively (obtained by subdividing the elution solutions mentioned above to equal volumes of 250 ml each) are combined according to comparative thin layer chromatography profiles, thus obtaining five distinct fractions. Said fractions have been denominated as Bark Extract-combination of 1-3 sub fractions of Hexane fraction (HMBal), Bark Extract- combination of 5-15 sub fractions of Hexane fraction (HMBa2), Bark Extract-combination of 16-79 sub fractions of Hexane fraction (HMBa3), Bark Extract-combination of 80-95 sub fractions of Hexane fraction (HMBa4), and Bark Extract-combination of 96-132 sub fractions of Hexane fraction (HMBa5). As it is discovered that the Bark Extract-combination of 16-79 sub fractions of Hexane fraction (HMBa3) features the best antibacterial effect, said fraction is purified using the silica gel column chromatography (Table 1). Graduated mixture of hexane-acetone ( The elution process was initiated using less potent solvent in the graduated elution, and the resolution at the beginning of the chromatogram was improved) is used for such purification process, and 100 different fractions of 100 ml are obtained as a result. Such fractions are: Hex-acetone (95:5; sub-fraction 1-27); Hex-acetone (90: 10; sub-fraction s 28- 46); Hex-acetone (85: 15; sub-fraction 47-55); Hex-acetone (80:20; sub-fraction 56-67); Hex- acetone (75:25; sub-fraction 68-72); Hex-acetone (70:30; sub-fraction 73-76); Hex-acetone (50:50; sub-fraction 77-84); Hex-acetone (30:70; sub-fraction 85-100); and acetone (100%; sub-fraction 101-103). Fractions no. 14-15 contains 15 mg FGA. Ferruginin A (FGA) orange oil; ¹³ C NMR (400 MHz, DMSO-de) d 192.8 (C-l), 105.9 (C-2), 180.7 (C-3), 51.3 (C-4),

142.6 (C-5), 116.2 (C-6), 137.9 (C-7), 124.5 (C-8), 140.8 (C-9), l23.5(C-lO), 155.8 (C-l l),

109.7 (C-l 2), 165.2 (C-13), 112.7 (C-14), 42.3 (C-15, C-15’), 120.2 (C-16, C-16’), 135.2 (C- 17, C-17’), 26.6 (C-l 8, C-18’), 21.6 (C-19 and C-19’), 26.3 (C-20), 119.9 (C-21), 132.0 (C- 22), 26.6 (C-23), 21.6 (C-24), 19.8 (C-25) [1] Table 1. Illustration of Fraction compositions.

The effect of FGA on expression of the MAPK genes in MCF-7 cells, the RT ² Profiler™ PCR Array Human MAP Kinase Signaling Pathway (PAHS-061A) was used based on the protocol provided by the producer of said kit (Qiagen, Hilden, Germany). The genes, which increase or decrease through application FGA, are indicated in Table 2, and illustrated in Figure 2. Accordingly, genes 12 and 34 increased and decreased two-fold, respectively. The gene that presents the highest increase is CDKN2B by 2.52-fold increase. The genes that present the highest decrease, on the other hand, are CDK4, CDKN1B, CREB1, KRAS, TP53 (8-fold), ATF2 ve B2M (l0.88-fold), CCNA2, CCNB1 (l2.7-fold) and ACTB (25.4-fold). Cycline- dependent kinase 4 inhibitor B, also known as multiple tumor suppressor 2 (MTS-2) or pl5INK4B, is coded with CDKN2B gene at the human beings. Said gene codes a Cycline- dependent kinase inhibitor that forms a complex with CDK4 or CDK6, and inhibits the cycline-dependent kinases to activate with cycline D. Therefore, coded protein functions as cell growth regulator and inhibits the cell cycle to proceed to Gl .

Table 2. The gene with expression that varies the most by application of Ferruginin A is the MAPK pathway genes.

Based on the control group, the gene with mRNA transcription (gene expression) reduced the most by FGA is the beta-actine (ACTB). B-actine mRNA reduces 25-fold compared to the control group. This gene codes one of the two proteins out of the actines of the cytoskeleton that is not associated with the muscle. The fact that the actines are one of the highly protected proteins as associated with the cell movement, structure and integrity indicates that the decrease at the expression of this gene is associated with apoptosis. Other genes with expressions reducing at significant levels are CCNA2 and CCNB1 genes. Cy cline- A2 is coded in human beings with CCNA2 gene [2] Cycline A2 is a member of the cycline family. The members of this family interact with CDK kinases and regulate progression of the cell cycle. Cycline-A2 distinguishes from other cycline-sugars with its ability to activate two distinct CDK kinases. While S phase bonds to CDK2, it then bonds to CDK1 during transition from G2 to M [3] Its transcription starts in late Gl phase and achieves its peak and saturation at the mid-S phase [4] Its excessive expression is observed in breast, cervix, liver and lung cancers [5, 6] Therefore, reduction in expression of this protein might be associated with the FGA mediated necrosis. G2/mitotic-specific cycline-Bl protein, on the other hand, is coded at the human beings with CCNB1 gene. Cycline Bl is a regulatory protein in mitosis. Therefore, reduction at the level of mRNA encoding a protein with regulatory role in mitosis might be associated with the FGA mediated necrosis. FGA and doxorubicin (Sigma-aldrich, St. Louis, MO, USA) cytotoxicity has been identified using neutral red test as defined before [7] As determined as a result of the application carried out for 48 and 72 hours according to US NCI scan program, a compound with ICso value less than 4 pg/mL features a good in vitro cytotoxic activity. FGA produced ICso values that are either lower or equal to the said value in all tested cancer cell lines. What’s interesting is that the IC50 values of FGA at SPC212 (0.20 pg/mL), the lung carcinoma cells, and MCF-7 (0.19 pg/mL), the breast carcinoma cells, are below 1 pg/mL. It is observed that FGA has low impact on the normal fibroblast CRL2120 cells, and presents selective toxicity by virtue of the selectivity index more than 3-fold (Table 3).

Table 3. Presentation of the cytotoxicity of Ferruginin A and doxorubicin on different cancer cell lines.

(*): The selectivity index is obtained by dividing normal IC50 value of the CRL2120 cells to IC50 value of the cancer cell line.

Wherein;

· A549: Human lung cancer cell line

• HepG2: Human liver cancer cell line

• SPC212: Human mesothelioma cell line

• DLD1 : Human colon cancer cell line

• Caco-2: Human colon cancer cell line

· MCF7 : Human lung cancer cell line

• CRL2120: Human normal skin cell line

The effect of FGA on the cell cycle and DNA content at MCF-7 cells after application for 72 hours has been analyzed using BD FACVerse™ System at the flow cytometry based on the directives provided by the producer of BD Cyclotest™ Plus DNA Reagent kit. It is observed that FGA triggers apoptosis depending on the dose and that it immobilizes the cell cycle at GO/G1 phase. The apoptotic cell ratio, which is 3.1% in the control group, reaches up to 19.8%; 16.4% and 12.8% after application of FGA for 72 hours at concentrations of 0.19 pg/mL (IC50), 0.10 pg/mL (½ x IC50) and 0.05 pg/mL (¼ x IC50), respectively.

In determination of the effect of FGA application in MCF-7 cells for 72 hours on MMP, the analysis is performed using flow cytometry according to the instructions for use of 5, 5', 6,6'- tetrachloro-1 ,l',3,3'-tetraethylhenzimidazo!ylcarhocyanine iodide (JC-l; Biomol, Germany) staining test kit (BD™ MitoScreen Mitochondrial Membrane Potential Kit). When compared to the control group, it is observed that application of FGA induces loss of MMP at MCF-7 cells, and that the loss of MMP, which is 4.3% at the control group, reaches up to 15.8% after application of FGA for 72 hours at a concentration of 0.19 pg/mL (IC50). This indicates that apoptosis triggered at MCF-7 cells with the impact of FGA occurs due to loss of MMP. (Figure 4)

Figure 5 illustrates the activity of caspase 3/7 and caspase 9 enzymes achieved as a result of applying Ferruginin A to MCF-7 cells for 6 hours through use of Caspase-Glo Assay kit (Promega, Mannheim, Germany) according to the manufacturer’s instructions for use for the kit. This indicates that FGA doesn’t trigger caspase 3/7 and caspase 9 activities during apoptosis.

In order to determine the impact of FGA on ROS production in MCF-7 cells, fluorescence analysis has been performed in Molecular Device SpectraMax M2 Absorbance/Fluorescence Microplate Reader through 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma-Aldrich, Germany) staining using the protocol provided by the manufacturer. It is seen that FGA allows an increase of ROS by more than 4 times (at IC50 value) in MCF-7 cells, thus triggering a significant level of ROS production. This indicates that apoptosis triggered at MCF-7 cells with the impact of FGA occurs due to activation of ROS production. Monitoring the inducing of the reactive oxygen species (ROS) level is important with respect to identification of the pathway for any drug candidate that triggers apoptosis. It is aimed to demonstrate under the scope of the invention the fact that FGA enhances the ROS level in the cell, thus impelling the cell to apoptosis. Here, the ROS level at IC50 concentration and the ROS level at ½ IC50, that is half IC50 concentration, has been determined and presented in graphical form (Figure 6). IC50: IC50 represents the drug concentration that causes the cell quantity to reduce by half. REFERENCES

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