Plant detected in the first fractions F5 to F10

Plant material collection:

Tabebuia rosea was collected in March 2016 from El-Orman garden, Giza, Egypt. The plant was authenticated by Dr. Mahmoud Elgebaly, Professor of Taxonomy, National Research center, Dokki, Egypt.

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Isolation and identification of endophytic fungi:

The protocol used for fungi isolation here is derived from (Janzo’s protocol)(Janso & Carter, 2010), and  it was modified according to the specific nature of plant tissue preliminary studies. The leaves were washed in tap water for one hour and cut into ten segments 5mm pieces. This was followed by applying the triple surface sterilization technique. Each piece was then placed on potato dextrose agar PDA (25g/l ) supplemented with Garamycin sulphate (40mg/l) at the rate of three to five pieces  per Petri plate and incubated  at room temperature 25-27 C for five days, which favors fungi growth. Further purification of the growing fungi mycelia was performed by subsequent sub-culturing on PDA (25g/l) until pure strains were separated. Identification was based on morphological characters of conidia and image analysis system in the data base identification program of the Regional Center for Mycology and Biotechnology (RCMB).

The endophytic strain from leaves codified as TR-S1 was stored as glycerol stocks ( 60% glycerol) in -80C in department of pharmacology, Ain Shams University.

                               

 

 

 

 

 

Aspergillus terreus

 

Fungal Cultivation and Extraction:

Agar blocks of TR-S1 endophyte were cut from the pure strain actively growing PDA Petri dishes and transferred to 1 L Erlenmeyer flasks containing 200 g sized recipe of Corn Grit Agar(CGA). TR-S1 endophyte was incubated at room temperature 26-28C for 21 days. Fungal metabolites were then extracted by acetone which was then partitioned with ethyl acetate and methanol respectively. TLC analysis of the ethyl acetate fraction revealed the characteristic blue spots of polyketides with vanillin/sulfuric spraying reagent as well as the positive results with Dragendorff’s reagent. Using silica gel column chromatography run with dichloromethane and methanol, Pulchranin A was detected in the first fractions F5 to F10 with Rf value of 0.25 in DCM: methanol 9.8:0.2 system and was isolated using preparative silica TLC with a yield value of 50 mg per 200g CGA culture.

 

Chemistry of Pulchranin A (Guzii et al., 2013):

?H(400MHz), ?C (

 

DMSO

CHCl3

Position

?H

?C

COSY

HMBC
 

?H

?C

1

 

153.67

 

 

 

 

NH

7.49(t, J=9.6)

 

H2

C3

 

 

NH2

7.47 brs
6.78 brs

 

 

 

 

 

2

3.73(dd)

53.2

NH 
H3

C1

3.52(s)

50.10

3

1.95(q, J=7.0)

31.96

 

C5

2.36

33.53

4

5.39 (dt,J=7.0, 15.5

129.20

H5, H3

 

5.38

129.69

5

5.55 (dt, J=7.0,15.5)

131.27

H4, H6

C7

5.32

131.91

6

1.96 (m)

38.96

H5, H7

 

2.07

27.39

7

3.51 (m)

69.37

H6

 

3.52

67.06

OH

3.95 brs

 

H7

 

 

 

8

1.25(m)

31.75

 

 

 

 

9

1.24(m)

29.52

H7

 

 

 

10

1.24(m)

29.47

 

C13 C12

 

 

11

1.25(m)

29.14  

 

 

 

 

12

1.29(m)

31.64

 

C10

 

 

13

1.26(m)

22.57

H14

C11, C14

 

 

14

0.85(t, 7.0)

14.45

H13

C12 C13

 

 

 

 

1H NMR (400 MHz, DMSO-d6) ? 7.48 (t, J = 9.6 Hz, 1H), 7.47( brs), 6.78 ( brs), 5.39 (d, J = 7.0, 15.5 Hz, 2H), 5.56(d, J=7.0, 15.5 Hz,2H),  3.50 (s, 0H), 3.73 (dd), 1.95 (q, J=7.0), 1.96 (m), 3.95 (brs), 1.29-1.24 (m, 12H), 0.86 (d, J = 7.0 Hz, 3H).

1H NMR (400 MHz, Chloroform-d) ? 5.38 (d, J = 6.4 Hz, 1H), 5.32 (s, 0H), 4.41 (s, 0H), 3.51 (s, 0H), 3.01 (s, 0H), 2.74 (s, 0H), 2.65 (s, 2H), 2.37 (s, 1H), 2.07 (d, J = 7.0 Hz, 1H), 1.78 – 1.56 (m, 2H), 1.34 (s, 1H), 1.28 (s, 4H), 0.91 (d, J = 3.8 Hz, 1H).

Pulchranin A gave M+H+ ion of m/z 256.2344 (HR-??????) with a Molecular formula of C13H3N3O. 1H and 13C NMR data (DMSO-d6, Table 1) revealed the presence of a guanidine group (dC 153.67; dH 6.87, 7.28, and 7.42), an N-substituted CH2 carbon (dC 53.2; dH 3.73), one terminal methyl (dH 0.86; dC 14.0), a double bond (dH 5.39 and 5.55; dC 129.20 and 131.27), an oxygenated methane (dH 3.51; dC 69.37), an OH group (dH 3.50) and nine methylene groups  .The 13C NMR and 2D-NMR spectra of 1 showed it to be an open-chain guanidine derivative with a double bond at C4–C5. The E geometry of the double bond was determined from the H-4/H-5 coupling constant of 15.4 Hz. The position of the hydroxy group was established using COSY correlations, marked as bold in Figure 1, including cross peaks between the signals of the hydroxy group proton and the methine proton at C-7.

 

 

 

 

Antimicrobial susceptibility assay:

Tested microorganism/sample code

TRS1(5mg/ml)

P3(6mg/ml)

control

Fungi

 

 

Amphotericin B

Aspergillus fumigatus
(RCMB 002008)

NA

NA

23

Candida albicans (RCMB 05036)

NA

NA

25

Penicillium aurantiogriseum (RCMB 001002)

NA

NA

20

Syncephalastrum racemosum (RCMB 016001)

20

20

22

Gram positive Bacteria

 

 

Ampicillin

Staphelococcus aureus (RCMB010010)

NA

NA

23

Bacillus subtilis (RCMB 010067)

10

14

32

Gram negative bacteria

 

 

Gentamycin

Salmonella sp. (RCMB010043)

17

16

17

Escherichia coli (RCMB010052)

9

11

19

 

Discussion:

Proliferation abnormalities which affect the cell cycle is an important feature of cancer initiation. Cyclin dependent kinases are activated in G1, S, G2 or M phase by the concomitant phosphorylation, activation of cyclin subunits or binding of activators &/ inhibitors subunits.  Cdk1, Cdk2, Cdk4 and Cdk6 are the most influential elements that contributes to both the normal and the human cancer cell cycle proliferation (Shapiro, 2006). Cdk4 and Cdk6 are responsible for the initiation of G1 phase, which is followed by the duplication of DNA for the S-phase. After the Cdk4/Cyclin D, Cdk6/cyclin D and the Cdk2/cyclin E complexes phosphorylate the retinoblastoma (Rb) proteins in the serine/threonine  residues, the Rb protein enhances the G1 phase(Franziska Graf, Wuest, & Pietzsch, 2009).

Before the restriction point is passed at the end of G1 phase, INK, Cip, Kip and other proteins may exert an inhibitory effect on Cdk2, Cdk4, Cdk6-cyclin complexes which modifies the Cdk catalytic activity. Consequently, this is followed by the formation of Cdk2-cyclin A complex that starts the S-phase events. While DNA damage checkpoint is mediated by the Cdk1-cyclin A, the G2/M phase transition is activated by the Cdk1-cyclin B. Therefore, active Cdk-cyclin complexes are the key to the activation of a myriad of substrates in the cell cycle, and if any core checkpoints are ignored, it leads to tumor cell progression and aberrant cell proliferation.

Here we investigate the effect of Pulchranin A, previously reported to induce JNK1/2, suppress AP-1 and induce p-53 cell death in JB6 Cl41 cells, on the viability of three different tumor cell lines MCF-7, HEP-G2 and HCT using Sulphorodamine Stain (SRB) assay, which showed IC50 values of 63, 80 and 91 µg/ml respectively. This is a moderate cytotoxic activity compared to the standard Doxorubin which gave 5.87 µg/ml.

Further investigations on the Breast cancer cell line MCF-7 revealed Pulchranin A role as an inhibitor of CdK1, Cdk2 and CdK4 where the IC50 measured for each of them was 9.82, 15.6 and 2.7 µg/ml. Compared to the standard Roscovitine with IC50 1.95, 5.03 and 1.4 µg/ml, Pulchranin A was moderately inhibiting cyclin dependent kinases. These results mean that Pulchranin A is promoting the pro-apoptotic pathway also in the Human Breast cancer cells MCF-7 through inhibiting the cyclin-dependent kinases in a dose dependent manner. As a result, it is interfering with the cell cycle check points in the G2/M phase as well as G1/S transition, which are both regulated by two axes: Rb protein and the cyclin-dependent kinases; however, it is not interfering with the initial re-entrance of the cell from G0 to G1 phase because it is not active against Cdk3, as it is evident from our results.

The interference of Pulchranin A with cyclin dependent kinases Cdk4 in Rb positive cells by blocking the ATP-binding site (Asp 145) confers its disruption of the Rb protein phosphorylation; therefore, activating its growth suppression properties and ending the advantageous growth features of the tumor cells over the normal ones. Moreover, blocking the ATP-binding site of Cdk2 by Pulchranin A is especially important in Rb negative or positive tumor cells because Cdk2 inhibition induces Rb-independent G1 cell cycle arrest.  As soon as the cell passes the restriction point at the end of G1 phase, Cdk2/cyclin A complex is formed, as it is responsible for the adequate performance of the cell in the S-phase. That is to say, DNA replication and phosphorylation of various proteins. In the G2 phase, through the inhibition of Cdk1/cyclin A complex, Pulchranin A can inactivate the DNA damage checkpoint control; thus, allowing malformed and aberrant DNA to progress in the cell cycle and eventually leading to cell death.

The abundant source of Pulchranin A, isolated for the first time from endophytes, provides an important amble source of this potentially non-genotoxic active molecule and puts it forward to be investigated for in-vivo and clinical trials experiments.

(F Graf, Mosch, Koehler, Bergmann, & Pietzsch, 2010)

 

Material and Methods:

Quantitative assay of Cyclin Dependent Kinases:

Cyclin dependent kinases (CDKs 1, 2, 3&4) were quantified using Elisa kits (Abcam, MA, USA) per the manufacturer’s instructions. Briefly, MCF-7 cells were seeded in 6 well cell culture plate in a density of 2×105 Cells/well and were left to attach overnight. Cells then were treated for 24 hours with the IC50 of the newly synthesized drug or left untreated (control). Following treatment cells were washed with phosphate buffered saline (PBS) three times and collected by trypsinization. Whole Cellular protein then were extracted by homogenization in the provided extraction buffer and equal protein quantities were used for the assay. Samples were added to the wells in the 96 well Elisa plate and were kept at 4 ? with continuous shaking for 30 min to allow binding of the protein to its primary antibodies. After washing, appropriate anti-CDK was added and the plate kept at 4 ? for 30 min with continuous shaking. Here after the plates were washed three times with PBS and TMB solution was added. After 30 min incubation stop reagent was added and the developed color was measured using Robonic P 2000 Elisa reader ( Robonic PVT Ltd., India) at 450 nM and the levels were calculated from the standard curve.

 

Measurement of potential cytotoxicity by SRB assay:(Skehan et al., 1990) (Dyshlovoy et al., 2016)

Cells were allowed to attach to the wall of the 96-multiwell plate for 24hrs at a concentration of 104cells/well, then the compound is added in different concentrations P3 (0, 1, 2.5, 5 and 10 µg/ml) to the cell monolayer. The test was performed in triplicate wells for each individual dose.

The compound was incubated with the cells for 48hrs at 37C and5% CO2.

Before staining with Sulforhodamine B stain, the cells are fixed, washed and stained. Any excess stains is removed by acetic acid leaving the attached one to be recovered by Tris EDTA buffer; consequently, the developed colour is measured by ELISA reader.

The survival curve for each tumor cell line was plotted after treatment with the P3.

 

Antimicrobial susceptibility assay:

The Susceptibility Tests were  performed according to NCCLS recommendations          ( National Committee for clinical  laboratory  Standards ,1993). Screening tests  regarding  the inhibition zone were carried out by the well diffusion method (Hindler et al., 1994). The inoculum suspension was prepared from colonies grown overnight  on an agar plate, and inoculated into Mueller-Hinton broth (fungi using malt broth)  . A sterile swab was immersed in the suspension and used to inoculate  Mueller-Hinton agar plates ( fungi using malt agar plates ). The compound were dissolved in dimethyl sulfoxide (DMSO) with different concentrations (10,5,2.5 mg/ml). the inhibition zone was measured around each well after 24h at 37Co. DMSO controls were adequately done.

Abstract:

The paucity of resources for natural anticancer drugs has been the topic of research in the last decade due to the challenges in obtaining them either from marine organisms or terrestrial plants. Recently, the microbial factories emerged as a promising source to produce a wide variety of bioactive natural products. Here we report for the first time the isolation and structure elucidation of Pulchranin A from Aspergillus terreus endophyte, which is cultured from the stem of Tabebuia rosea terrestrial plant. Using    docking                                , it is shown that Pulchranin A has a good interaction with Cdk1, Cdk2 and Cdk4 with……………………..This is followed by invitro studies for its anticancer activity using SRB assay in MCF-7, Hep-G2 and HCT cell lines, which showed a promising cytotoxic activity with IC50 63, 80 and 91 µg/ml respectively. By assaying the cyclin dependant kinases in MCF-7 cells, Pulchranin A inhibited Cdk1, Cd2 and Cdk4 with IC50   9.82, 15.6 and 2.7 µg/ml respectively suggesting a potent non-genotoxic Cdk inhibitor for the breast cancer cells.