Replication Of Dna Influenced Topological State Genetic Materia Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

The replication of DNA is influenced by the topological state of the genetic material.1 Topoisomerase is such kind of enzyme which can relieve any torsional stress that develops in cellular DNA molecules during replication. So a number of nucleic acid metabolism, such as replication, transcription, chromatin remodeling, recombination and repair are be modified by the enzymes. This kind of enzyme are distinguished by their catalytic mechanisms. The Topo I acts by generating a transient single-stranded break in the double helix, while the Topo II acts by generating a transient double-stranded break. Furthermore, the Topo II is ATP dependent. 2 -5

In 1960s, Monroe Wall and co-workers found the crude extracts of Camptotheca acuminate were quite active in the L1210 mouse leukemia life prolongation assay among hundreds of plant extracts been tested.6 Camptothecin (CPT) was identified as most active compound, and the sodium salt of which was tested clinically in the mid 1970s, but was discontinued because of its serious side effects. Until the Topo I was discovered as its cellular targets, the water-soluble derivatives of CPT, topotecan and irinotecan were successfully developed and used in clinic to treat ovarian cancer and colon cancer respectively.7 The mechanism of CPT was demonstrated as Topo I poison, which stabilize the covalent Topo I-DNA cleavage complex forming in the transesterification reactions to break and rejoin DNA strand, Hence, reduction of cleavage complexes leads to reduced cell death. Two mechanisms were reported for a reduction of the Topo I cleavage complexes: (1) Topo I mutations that attribute to the enzyme drug resistant, and (2) decrease topo I activity.8 Due to the clinic limit of CPT and the realization of its unique mechanism, many non-CPT derivatives has been synthesized, like indolocarzazoles and indenoisoquinolines, some of which are now in clinical evaluation.9 Furthermore, several Topo II poison like amsacrine (m-AMSA) and etoposide,10, 11 which stabilize the covalent Topo II-DNA cleavage complex on both DNA strands, quinolone antibiotics acting as DNA gyrase inhibitors12 and a small class of compounds inhibiting both Topo I and Topo II, including aclarubicin, intopolincine and some indenoisoquinolinones were developed and many of which have been used in clinic.13 - 15

Compared with the topoisomrease poisons, the compounds interfering any of the other steps in the catalytic cycle are named catalytic inhibitors, which act at a step upstream of DNA cleavage and covalent binding to DNA.16, 17 The topoisomerase catalytic inhibitors do not generate DNA strand breaks and should be more active in cells with low topoisomerase levels. The catalytic Topo II inhibitors contain a variety of compounds that might interfere with the binding between DNA and topoisomerase, stabilize noncovalent DNA topoisomerase complex or inhibit Topo II-ATP binding.17

In an effort to identify quinolone alkaloids as topoisomerase inhibitors from the Evodia rutaecarpa and to understand their mechanism of action, eight alkaloids were isolated, two of which were quinazolinocarboline alkaloids, evodiamine and retaecarpine, and the other six compounds were quinolone alkaloids, and their inhibitory activities against topoisomerase were evaluated. We found three of them exhibited potent inhibitory activities against human topoisomerase. Among them, evodiamine, which is the most active in primary screen, showed cytotoxicity against human leukaemia K562 and THP-1 cell lines with IC50 values of 34.35 μM and 90.87 μM respectively. Importantly, this is the first time that the mechanism of evodiamine was demonstrated as catalytic inhibitor of both Topo I and Topo II, since this compound did not show any evidence for the formation of the enzyme-DNA cleavage complex. However, the effect of evodiamine on the individual step of the catalytic cycle is still not fully understood.

Results

Isolation and identification of quinolone alkaloids from Evodia rutaecarpa

The Evodia crude extract were separated according to the literature18 with gradient system of methanol, acetonitrile and water. Eight fractions were collected using reverse-phase HPLC with Waters semi-preparative column (7.8 mm x 300 mm, 7 μm) detected at  254 nm, and the structures of them were elucidated by 1-D, 2-D NMR and Mass spectroscopic methods. Although these compounds have been known for some years and the similar chromatograph obtained using HPLC method one years before25, we got the different view with the structure of 6, which was identified as evocarpine by all the spectroscopic method in our study, since two olifinic protons at δH 5.35 correlating to two carbons at δC 129.94 were assigned to alkene chain (data not shown).

Table 1. Structures of alkaloids isolated from Evodia rutaecarpa using semi-preparative reverse-phase HPLC

1

2

R

3

4

5

6

7

8

Antiproliferation study against human leukaemia cell lines

The MTT-based assay was used to evaluate the antiproliferative activities against THP-1 cell line for compounds (1 - 8) and m-AMSA. The viabilities of THP-1 cells for 5 d continuously treated were shown in Figure 1, from which, 1 and the positive control m-AMSA exhibited antiproliferative activities against THP-1 cell line among all the compounds.

Figure 1. Effect of compounds (1 - 8 and m-AMSA) with concentrations of 5 ng/μL on the antiproliferation of THP-1 cell line

Consequently, the IC50 values of 1 on the antiproliferation activity against human leukemia THP-1 and K562 cell lines were compared with two anti-tumor drugs, m-AMSA and etoposide for 1 h treatment. As shown in Figure 2, evodiamine (1) was more effective against K562 cell line with an IC50 value of 34.35 μM, which was better than the topoisomerase II poison etoposide with an IC50 value of 53.26 μM. Evodiamine (1), a characteristic quinazolinocarbolin alkaloid from Evodia, has been reported to inhibit the invasion and metastasis of tumors, and induces cell death in several types of cancer cells, such as human acute leukemia CCRF-CEM cells (IC50 0.57 µM)26, human androgen independent prostate cancer PC-3 cells (IC50 1.53 µM)27, human breast cancer MCF-7 cells (IC50 6.02 µM)28, human melanoma A375-S2 cells (IC50 15 µM)29 and murine fibrosarcoma L929 cells (IC50 20.3 µM)30. It possessed lowest cytotoxicity against THP-1 cells among all the reported cell lines with an IC50 value of 90.87 μM.

Figure 2. IC50

Effects of quinolone alkaloids on topoisomerase activity

As the mechanisms of evodiamine have been demonstrated to enhance the polymerized tubulin levels26 and stabilize the Topo I -DNA cleavage complex as a Topo I poison28, to investigate the mechanisms by which the quinolone alkaloids inhibited either topo I or topo II and thereby caused cytotoxicity, the effect of all compounds on topoisomerase activity were examined by measuring the relaxation of supercoiled DNA of plasmid pBR 322 for Topo I in the absence or presence ethidium bromide (EtBr) and decatanation of kinetoplase DNA for Topo II. The gels without EtBr allow the detection of compounds inhibiting DNA relaxation by Topo I, while EtBr containing gels indicate whether or not the inhibiting activities of those compounds might be due to the effects on the Topo I-DNA cleavage complexes. Compounds 1, 3 and 7 exhibited Topo I inhibitory activities (Figure 3A), of which, 1 was the most effective, since the relaxation of supercoiled DNA was totally inhibited incubating with 100 μM of 1. Compared with 1, CPT did not unwind DNA to any detectable extent up to 100 μM, while it increased the intensity of nicked band (Figure 3B, lane 3), which indicated the formation of the enzyme-DNA cleavage complex with one strand of DNA broken. Furthermore, 1 did not show any evidence increasing the intensity of nicked band in the EtBr containing gel (Figure 3B, lane 4) and thus 1 might be not acting as Topo I poison.

Compounds 1 and 7 possessed Topo II inhibitory activities (Figure 3C), of which, 1 (evodiamine) showed more effective inhibitory activity compared with the Topo II poison m-AMSA as 90% of catanated kDNA remained in the well (Figure 3C, lane 4) when it incubated with 100 μM of 1 (evodiamine), while only 17.8% of catanated kDNA was in the well after incubating with 100 μM of m-AMSA (Figure 3C, lane 3) which was similar inhibitory activity with 7. However, 7 did not show any cytotoxocity against two tested leukemia cell lines (data not shown).

A

B

C

Figure 3. Effect of compounds (1 - 7) with concentration of 100 µM on the relaxation of plasmid pBR 322 DNA (0.5 µg) by 1 U of human Topo I. DNA samples were separated by electrophoresis on the 1% agarose gel (A) with or (B) without ethidium bromide. The gels were photographed under UV light. Nck, nicked; Rel, relaxed; Sc, supercoiled. (C) Effect of 100 µM of compounds (1 - 7) on the decatanation of kDNA (0.2µg) by 1 U of human Topo II.

The inhibitory activities of human Topo I and Topo II by evodiamine (1) were further evaluated by Topo I relaxation assay and Topo II decatanation assay respectively in concentration-dependant manner. The IC50 values of evodiamine (1) against human Topo I and Topo II were 60.74 μM and 78.81 µM respectively. Thus, evodiamine (1) was identified as a dual inhibitor against both Topo I and Topo II. More recently, a number of drugs have been identified that target both Topo I and Topo II, and some of these drugs have been advanced to clinical trial. They can act as poison-poison (Intoplicine), poison-catalytic inhibitor (Aclarubicine) or catalytic-catalytic inhibitors (F11782).31 - 33 The mechanistic studies were carried out to investigate which type of inhibitors the 1 acting as.

A

C

B

D

Figure 4. IC50 evaluation of evodiamine (1) inhibitory activities against human Topo I and Topo II

Effect of 1 on the Topo II-mediated DNA cleavage assay

Topoisomerase II poisons such as AMSA are known to stabilize the cleavage complex that leads to DNA strand breaks, while catalytic inhibitors such as aclarubicin can protect cells against Topo II poison-induced DNA damage. As shown in the previous results, 1 is a Topo I catalytic inhibitor. We also tested whether 1 could perform as a poison and stimulate the DNA cleavage complexes of Topo II using the Topo II cleavage assay. In contrast to m-AMSA, which stimulated the cleavage complex indicating as linear band in the gel, 1 had no effect on Topo II cleavage activity even the concentration up to 100 µM and Topo II activity was partially inhibited incubating with thus high concentration of enzyme (10 U).

Figure 5. 1 did not induce the accumulation of the Topo II-DNA cleavage complex. Supercoiled pBR 322 DNA (0.3 µg) (lane 1) was incubated with 10 U of human topo II in absence (lane 2) or presence of AMSA or 1. Oc, Open circlar. Lin, Linear DNA.

Interaction between 1 and DNA

The drug-free DNA sample is negatively supercoiled and migrates through down the gel as a single band. But its profile changes significantly in the presence of intercalating agents. At low concentration, the DNA intercalation of the agent between DNA base pairs induces the relaxation of DNA, which shows slowly migrates on the top of the gel. As the concentration further increased, the DNA molecules wind in the oppposite way so as to generate positive supercoils. When the DNA is fully positively supercoiled, it migrate as a single band with an mobility close to that of the negatively supercoiled DNA. Therefore, the typical intercalating agent should show a up-and-down profile with increasing concentration.

As indicated in Figure 6A, the DNA intercalator EtBr showed clearly the up-and-down profile with the increasing concentration when incubated with 10 U of human Topo I, while CPT and 1 did not effect the assay. DNase I footprinting (Figure 6B) indicated 1 did not bind to DNA at the concentration up to 100 µM. The two Topo II poison, etoposide and AMSA did not bind to DNA at the concentration of 100 µM, since etoposide was identified as non-DNA binder and AMSA is DNA intercalator.

A

B

Figure 6. (A) Supercoiled pBR 322 DNA (0.3 µg) (lane 1) was incubated with 10 units of human topoisomerase I in absence (lane 2) or presence of serial dilution of EtBr (lanes 3 to 7), CPT (lanes 8 to 11) or Evodiamine (lanes 12 to 16). (B) DNA footprinting showed evodiamine did not bind to DNA at the concentration up to 100 µM. Furthermore, the Topo II poison etoposide and m-AMSA did not bind to DNA, since etoposide is known as DNA non-binder and m-AMSA is DNA intercalater.

Effect of 1 on cell cycle distribution in K562 cell line

To investigate the effect of 1 on cell cycle progression, K562 cells were exposed to 1 for 1 h, and then grew in drug free medium until test. Cell cycle distribution was determined by flow cytometric analysis. As shown in Figure 7, 1 clearly induced G2/M arrest in exponentially cells treated by 20 μM of drug. G2/M arrest was demonstrated as effect of topoisomerase II inhibitors, such as etoposide.25

Figure 7. Cell cycle distribution analysis of K562 cells exposed to 20, 40 or 80 μM of 1. Cell were treated for 1 h, following grew in drug free medium for 8, 24, 32, 48 and 72 h before test.

We next evaluated the possibility that the cytotoxicity of 1 in K562 cells was not attributable to a higher level of induced DNA damage. The potential of 1 to induce DNA strand breaks in K562 cells was assessed via Comet assay using the Topo I poison CPT as a positive control. There is no any strand breaks induced by 1 up to 100 µM, while CPT treated K562 cells exhibited highly damaged in the assay (Figure 8). The Topo I poison CPT achieve their cytotoxicity effects by stabilizing the enzyme-DNA cleavage complex which leads to DNA strand breaks in chromosomal DNA and results in cell death. Consistent with the observation in Topo II-mediated DNA cleavage assay, 1 did not cause any DNA damage. So the mechanism of 1 inhibiting of topoisomerase was considered as both Topo I and Topo II catalytic inhibition, but not topoisomerase poison action.

CPT

Control

10 μM

1

Control

100 μM

Figure 8. The comet assay was applied to detect the DNA strand breaks inducing by evodiamine. The Topo I poison CPT showed highly DNA damaged cells at the concentration of 10 μM, while the DNA strand breaks were not detected for the evodiamine treated cells even at the concentration up to 100 μM.

Possible effects of 1 on the CPT resistant cell line

Topo I poisons, such as CPT, are cytotoxic to the cells by trapping cleavage complex rather than inhibiting enzyme catalytic activity, which indicated that they turn Topo I into a cellular poison that induce DNA damage. Therefore, the reduction of Topo I activity is one of the first change in cells selected for CPT resistance, and conversely, sensitivity of Topo I poisons increase with the enzyme overexpression.34, 35 On the other hand, Figure 5 indicated 1 is more efficient inhibitory activity incubating with lower concentration of enzyme and the sensitivity reduced with increasing of enzyme. Therefore, 1 might be effective on the CPT resistant cell lines.

Figure 9. 100 μM of evodiamine (lane 3 to 7) or camptothecin (lane 8 to 12) were incubated with serial dilution of human topoisomerase I and pBR 322 DNA (0.3 µg). DNA samples were separated by electrophoresis on the 1% agarose gel.

Discussion

Conclusion

Experimental Section

General Experimental Procedure

Chemistry

The methanol and acetonitrile used as mobile phase and mass spectrometry solvents were HPLC grade and punchers from Fisher Scientific (UK). Distilled water used in extraction and HPLC were filtered by Water Purify System (Millipore SimplicityTM, USA). All the other chemicals and solvent were laboratory grade and used without further purification.

Extraction and Isolation of Quinolone Alkaloids

The dry fruit of Evodia powder (ca. 50 g) was soaked in ethyl acetate (800 mL) for 30 min, and then extracted by ultrasonification for 45 min (the extraction procedure was repeated twice for each sample and four samples were extracted). The ethyl acetate layers were filtered and combined, following that, the solvent was evaporated by a rotary evaporator under reduced pressure to four give four brown crude extracts (1, 2, 3 and 4). After dissolving in methanol and kept at 4°C for 24 h, yellow crystals generated, which was identified as rutaecarpine by NMR spectroscopic method. The rutaecarpine crystals were separated out from the crude extract (1, 2 and 4), recrystallized by dichloromethane and weighted for yield. Then the extracts without rutaecarpine crystals were separated by reverse-phase HPLC from Jasco (Japan, consisting of PU-980 intelligent HPLC pumps, UV-975 intelligent UV/VIS detectors and LC-980-02 ternary gradient) with the separate method according to the literature [18] on a Waters semi-preparative column (7.8 mm x 300 mm, 7 μm). The separations were carried out using a gradient system of methanol, acetonitrile and water (0 min: 2%:38%:60%, 22 min: 5%:38%:57%, 35 min: 30%:38%:32% and 65min: 45%:38%:17%) with flow rate 4.5 mL/min. The detector wavelength was set at 254 nm.

HPTLC quantification of rutaecarpine in crude extract

Standard

The evodiamine and rutaecarpine standards were supplied by SUTCM (Shanghai, China) and a solution of a 1 mg/mL of each standard was prepared by dissolving the standard in chloroform. The crystal (XP-E-1) obtained from the evodiamine extract was dissolved in chloroform to give a 1 mg/mL solution.

Developing solvent

Hexane (14 mL) was mixed with chloroform (4 mL) and acetone (2 mL) to give a total volume of 20 mL developing solvent.

Derivatizing reagent

Sulfuric acid (20 mL) was gently added to ice cold methanol (180 mL), mixed carefully and allowed to cool down to room temperature.

HPTLC identification was carried out at room temperature using a CAMAG HPTLC system (CAMAG, Switzerland) including an automatic TLC sampler (ATS 4), automatic developing chamber (ADC 2), TLC visualizer (150904) and TLC software (winCATS). The glass plates, HPTLC silica gel 60 F254, 20 x 10 cm and electro-pneumatic TLC-sprayer were purchased from Merck (Merck, Germany).

Test solutions and standard solutions (2 µL) were applied with a band length of 8 mm each, 8 mm from the lower edge and 22 mm from left and right edges of the plates. The developing solvent (20 mL) were poured into an ADC 2 developing chamber and saturated for 15 min before development. The plate was dried before and after developing for 5 min. Migration distance was set at 80 mm. After developing, the un-derivatized plate was documented using UV 254 and 366 illuminations. Then the plate was sprayed with the derivatizing reagent, dried for 10 min and placed onto a preheated hot plate (105 oC) for 5 min. After cooling to room temperature, the plate was documented with white, 254 and 266 nm illuminations.

Characterization of Quinolone Alkaloids

NMR spectra were obtained using Bruker Avance 500 NMR Spectrometer (Bruker corp. UK). TMS (tetramethylsilane) is used as a reference for all NMR measurements. The pulse programs were Zg30 for normal 1H-NMR, Zgpg30 for normal 13C-NMR, and DEPT30 and DEPT90 for 13C DEPT spectra. The MS spectra were measured using a Waters Q-Tof microTM (Waters corp. USA) mass spectrometer.

Evodiamine (1): 1H NMR (500.13 MHz, CD3OD) δ: 2.52 (3H, s, N-CH3), 3.00 (2H, m, H-6), 3.32, 4.90 (1H, each, m, H-5), 5.95 (1H, s, H-3), 7.17 - 8.16 (8H, m, Ar-H), 8.22 (1H, br, N-H). 13C NMR (125.76 MHz, CD3OD) δ: 20.44 (C-6), 42.58 (C-5), 40.83 (N-CH3), 113.22 (C-12), 119.64 (C-7), 120.8596 (C-9), 120.23 (C-11), 122.07 (C-20), 126.56 (C-10), 126.88 (C-8), 127.45 (C-18), 127.47 (C-19), 127.99 (C-16), 128.32 (C-2), 135.95 (C-17), 140.55 (C-13), 146.80 (C-3), 149.31 (C-15), 163.46 (C-21). [M+H]+ m/z 304.1076. Compound EF-2 was identified as. [65]

Rutaecarpine (2): 1H NMR (500.13 MHz, CD3OD) δ: 3.26 (2H, t, H-6), 4.55 (2H, t, H-5), 7.12 (1H, t, H-10), 7.29 (1H, t, H-11), 7.45 (1H, m, H-18), 7.47 (1H, m, H-12), 7.64 (1H, d, H-9), 7.72 (1H, d, H-16), 7.79 (1H, dt, H-17), 8.23 (1H, dd, H-19), 8.54 (1H, br, N-H). 13C NMR (125.76 MHz, CD3OD) δ: 20.44 (C-6), 42.58 (C-5), 113.32 (C-12), 119.63 (C-7), 120.96 (C-9), 121.23 (C-11), 121.95 (C-20), 126.33 (C-10), 126.71 (C-8), 127.28 (C-18), 127.82 (C-19), 127.99 (C-16), 128.27 (C-2), 135.69 (C-17), 140.48 (C-13), 146.80 (C-3), 149.31 (C-15), 163.46 (C-21), [M+H]+ m/z 288.3462. Compound EF-3 was identified as rutaecarpien.[65]

1-methyl-2-nonyl-4(1H)-quinolone (3): 1H NMR (500.13 MHz, CDCl3) δ: 8.45 (1H, dd, H-5), 7.67 (1H, m, H-7), 7.51 (1H, d, H-8), 7.38 (1H, m, H-6), 6.25 (1H, s, H-3), 3.75 (3H, s, N-CH3), 2.72 (2H, t, H-1'), 1.69 (2H, m, H-2'), 1.43 (2H, m, H-3'), 1.37 - 1.26 (10H, m, H-4', H-5', H-6', H-7', H-8'), 0.88 (3H, t, H-9'). 13C NMR (125.76 MHz, CDCl3) δ: 177.86 (C-4), 154.86 (C-2), 141.95 (C-8a), 132.02 (C-7), 126.70 (C-5), 126.57 (C-4a), 123.29 (C-6), 115.29 (C-8), 111.23 (C-3), 34.81 (C-1'), 34.13 (N-CH3), 28.58 (C-2'), 31.89 - 22.69 (Alkyl chain), 14.14 (C-9'), [M+H]+ m/z 286.3147, EF-4 was identified as 1-methyl-2-nonyl-4(1H)-quinolone.[31]

1-methyl-2-[(Z)-5-undecenyl]-4(1H)-quinolone (4): 1H NMR (500.13 MHz, CDCl3) δ: 8.45 (1H, dd, H-5), 7.67 (1H, m, H-7), 7.51 (1H, d, H-8), 7.38 (1H, m, H-6), 6.25 (1H, s, H-3), 3.75 (3H, s, N-CH3), 2.72 (2H, t, H-1'), 1.69 (2H, m, H-2'), 1.43 (2H, m, H-3'), 1.37 - 1.26 (10H, m, H-4', H-5', H-6', H-7', H-8'), 0.88 (3H, t, H-11'). 13C NMR (125.76 MHz, CDCl3) δ: 177.86 (C-4), 154.86 (C-2), 141.95 (C-8a), 132.02 (C-7), 129.94 (Olefinic) 126.70 (C-5), 126.57 (C-4a), 123.29 (C-6), 115.29 (C-8), 111.23 (C-3), 34.81 (C-1'), 34.13 (N-CH3), 31.89 - 22.69 (Alkyl chain), 14.14 (C-11'), [M+H]+ m/z 313.2115, EF-5 was identified as 1-methyl-2-[(Z)-5-undecenyl]-4(1H)-quinolone.[31]

1-methyl-2-dodecyl-4(1H)-quinolone (5): 1H NMR (500.13 MHz, CDCl3) δ: 8.31 (1H, dd, H-5), 7.89 (1H, d, H-8), 7.80 (1H, m, H-7), 7.46 (1H, m, H-6), 6.32 (1H, s, H-3), 3.90 (3H, s, N-CH3), 2.89 (2H, t, H-1'), 1.73 (2H, m, H-2'), 1.50 (2H, m, H-3'), 1.46 - 1.31(12H, m, H-4', H-5', H-6', H-7', H-8', H-9'), 0.89 (3H, t, H-11'). 13C NMR (125.76 MHz, CDCl3) δ: 179.61 (C-4), 159.00 (C-2), 143.52 (C-8a), 133.95 (C-7), 127.00 (C-5), 126.69 (C-4a), 125.18 (C-6), 117.89 (C-8), 111.15 (C-3), 35.70 (C-1'), 35.49 (N-CH3), 31.89 - 22.69 (Alkyl chain), 14.47 (C-11'), [M+H]+ m/z 315.1296, EF-6 was identified as 1-methyl-2-dodecyl-4(1H)-quinolone. [31]

Evocarpine (6): 1H NMR (500.13 MHz, CDCl3) δ: 8.31 (1H, dd, H-5), 7.87 (1H, d, H-8), 7.80 (1H, m, H-7), 7.48 (1H, m, H-6), 6.32 (1H, s, H-3), 5.34 (2H, m, H-7' and H-8'), 3.90 (3H, s, N-CH3), 2.89 (2H, t, H-1'), 1.73 (2H, m, H-2'), 1.50 (2H, m, H-3'), 1.46 - 1.31(12H, m, H-4', H-5', H-6', H-9', H-10', H-11' and H-12'), 0.89 (3H, t, H-13'). 13C NMR (125.76 MHz, CDCl3) δ: 179.61 (C-4), 159.00 (C-2), 143.52 (C-8a), 133.95 (C-7), 130.9 and 130.7 (Olefinic), 127.00 (C-5), 126.69 (C-4a), 125.18 (C-6), 117.89 (C-8), 111.15 (C-3), 35.70 (C-1'), 35.49 (N-CH3), 32.89 - 21.40 (Alkyl chain),14.47 (C-13'), [M+H]+ m/z 340.8739, EF-7 was identified as evocarpine.[31]

1-methyl-2-[(6Z,9Z)]-6,9,-pentadecadienyl-4(1H)-quinolone (7): 1H NMR (500.13 MHz, CDCl3) δ: 8.31 (1H, dd, H-5), 7.87 (1H, d, H-8), 7.80 (1H, m, H-7), 7.48 (1H, m, H-6), 6.32 (1H, s, H-3), 5.34 (2H, m,), 3.90 (3H, s, N-CH3), 2.89 (2H, t, H-1'), 1.73 (2H, m, H-2'), 1.50 (2H, m, H-3'), 1.46 - 1.31(12H, m, H-4', H-5', H-6', H-7', H-8', H-9'), 0.89 (3H, t, H-15'). 13C NMR (125.76 MHz, CDCl3) δ: 179.61 (C-4), 159.00 (C-2), 143.52 (C-8a), 133.95 (C-7), 130.2 (Olefinic), 127.00 (C-5), 126.69 (C-4a), 125.18 (C-6), 117.89 (C-8), 111.15 (C-3), 37.6 (C-8'), 35.70 (C-1'), 35.49 (N-CH3), 29.73 - 24.11 (Alkyl chain), 14.47 (C-15') [M+H]+ m/z 366.9176, EF-8 was identified as 1-methyl-2-[(6Z,9Z)]-6,9,-pentadecadienyl-4(1H)-quinolone.[65]

Dihydroevocarpine (8): 1H NMR (500.13 0MHz, CD3OD) δ: 8.31 (1H, dd, H-5), 7.87 (1H, d, H-8), 7.80 (1H, m, H-7), 7.48 (1H, m, H-6), 6.32 (1H, s, H-3), 5.34 (2H, m,), 3.90 (3H, s, N-CH3), 2.89 (2H, t, H-1'), 1.73 (2H, m, H-2'), 1.50 (2H, m, H-3'), 1.46 - 1.31(12H, m, H-4', H-5', H-6', H-7', H-8', H-9'), 0.89 (3H, t, H-13'). 13C NMR (125.76 MHz, CD3OD) δ: 179.61 (C-4), 159.00 (C-2), 143.52 (C-8a), 133.95 (C-7), 127.00 (C-5), 126.69 (C-4a), 125.18 (C-6), 117.89 (C-8), 111.15 (C-3), 35.70 (C-1'), 35.49 (N-CH3), 31.89 - 22.69 (Alkyl chain), 14.47 (C-13'), [M+H]+ m/z 342.4131, EF-9 was identified as dihydroevocarpine.[31]

Materials and Methods for Biological Assay

Cell culture and cytotoxicity assay

Human K562 myelogenous leukemia cells and THP-1 acute monocytic leukemia cells were obtained from the American Type Culture Collection (ATCC, UK). Cells were grown at 37 °C in a humidified atmosphere containing 5% CO2 in RPMI-1640 medium with 10% of fetal bovine serum and 2 mM of glutamine. The viability of cell line were determined using the MTT [3-(4,5-dimethyl thiazol-2yl)-2,5-diphemyltetrazolium bromide [Sigma, UK] assay. 19, 20 2 mL of cells with the concentration 5 - 105 cells/mL (counted by hemocytometer) in exponential growth were treated with varying concentrations (0.3 to 100 μM) of compounds under study in duplicate for 1 h or 24 h. 1% DMSO treated cells were taken as negative control. Following treatment, cells were pelleted (1500 rpm, 5 min), resuspend in 2 mL of RPMI-1640 medium, plated out at 200 μL per well in U bottom plates and incubated for 96 h at 37°C. At the end of the incubation, 20 μL of MTT (5 mg/mL in distilled water) was added and incubated for 4 h. Formazan crystals formed were dissolved in 200 μL of DMSO. The absorbance was measured at 540 nm in the plate reader (Varioskan Flash, Thermo Scientific, UK). The IC50 values were estimated by plotting the data on Grafit 5.0.4 version (Erithacus Software, UK).

Topoisomerase I relaxation assay

Topoisomerase relaxation assay and decatanation assay were carried out as previously described.21, 22 Supercoiled pBR 322 DNA (0.3 µg) was incubated with 1 U of human Topo I (Inspiralis, UK) in 30 μL of relaxation buffer (2 mM Tris-HCl pH 7.5, 20 mM NaCl, 25 µM EDTA and 0.5% glycerol) at 37°C for 30 min in the presence of varying concentrations (0.3 to 100 μM) of the compounds under study. 100 µM of Topo I poison CPT was used as positive control.

5 U of the enzyme was used to study the intercalating ability of some compounds based on the Topo I relaxation assay.23

The ability of compounds to stabilize the covalent DNA enzyme reaction intermediate were evaluated by incubating supercoiled pBR 322 DNA (0.3 µg) with 10 U of human Topo II for the relaxation assay. After incubation with enzyme, the reaction was further incubated with 0.1 mg/mL proteinase K and 0.2% SDS for 30 min at 37°C.

Topoisomerase II decatanation assay

kDNA (200 ng) was incubated with 1 unit of human Topo II (Inspiralis, UK) in assay buffer (5 mM Tris-HCl pH 7.5, 12.5 mM NaCl, 1 mM MgCl2, 0.5 mM DTT and 10 μg/mL albumin) with 1 mM of ATP at 37 °C for 30 min in the presence of different concentrations (3 to 100 μM) of the compounds under study. The Topo II poisons, amsacrine and etoposide, were served as positive control.

Gel electrophoresis

The topoisomerase catalytic reactions were stopped by addition of 18 μL of stop buffer (40% sucrose, 100 mM Tris pH7.5, 10 mM disodium EDTA and 0.05% bromophenol blue). DNA samples were extracted by a mixture of Chloroform/iso amyl alcohol (24:1), followed by it running on a 1% agarose gel at 10 V /cm in TAE buffer for 3 h. Gels were stained with ethidium bromide (0.5 μg/mL in distilled water) for 1 h and destained with distilled water for 30 min. Similar experiments were performed using ethidium bromide (0.5 μg/mL) containing agarose gel to achieve separation of nicked and relaxed species. The gel was visualized under UV light and photographed. For quantitative determinations, the integrated intensities of the ethidium bromide fluorescence of the bands (supercoiled form for relaxation assay and minicircled form for decatanation assay) were quantified and calculated using ImageJ software (Wayne Rasband, USA). The IC50 values were estimated by plotting the data on Grafit 5.0.4 version (Erithacus Software, UK).

Comet assay

The single cell gel electrophoresis (comet) assay developed to allow visualization of DNA strand break damage in individual cells was performed as preciously described.24 2 mL of K562 cells with the concentration 2.5 - 104 cells/mL (counted by hemocytometer) in exponential growth were treated with various concentrations of compounds under study for 1 h. 1% DMSO treated cells were taken as negative control. 0.5 mL of cell suspension was mixed with 1 mL of pre-warmed low gelling temperature (LGT) agarose (1% water). 1 mL of the mixture was rapidly spread on the single-frosted glass microscope slid (25 - 75 mm, 1 mm thick) pre-coated with 1 mL of type 1-A agarose (1% water) and covered with a coverslip (24 - 40 mm). Then 500 mL of ice-cold lysis buffer (0.1 M disodium EDTA, 2.5 M NaCl, 10 mM Tris-HCl pH 10.5 with NaOH and 1% Triton X-100 freshly added before use) was added to submerge all slides and incubated on ice for 1 h in dark. The slides were washed by ice-cold double distilled water, fowllowed by it transferring into the electrophoresis tank with 2 L of ice-cold alkali buffer (50 mM NaOH, 1mM disodium EDTA pH 12.5) and incubated for 45 min in dark to allow unwinding of DNA. Electrophoresis (0.6 V/cm, 250 mA) was then carried out at room temperature for 18 min in dark. After electrophoresis, slides were washed with neutralisation buffer (0.5 M Tris-HCl, pH 7.5), left for 10 min and rinsed twice with PBS. After drying overnight at room temperature, slides were stained twice with 1 mL of propidium iodide solution (2.5 μg/mL). All slides were dried in oven at 37°C and stored until image analysis. 50 randomly selected individual cells per slides were visualised and the olive tail moments (OTM) were measured using the comet imaging system () with fluorescent microscope.

Cell cycle analysis

Flow cytometry analysis of DNA content, 2 mL of K562 cells with the concentration 5 - 105 cells/mL (counted by hemocytometer) in exponential growth were treated with different concentrations (20 μM, 40 μM and 80 μM) of compounds under study for 1 h and then resuspended in 2 mL of RPMI-1640 medium for time course (0 h, 8 h, 24 h, 32 h, 48 h and 72 h). At the end of each time course, cells were centrifuged (1500 rpm, 5 min) and the pellets were suspended in 1 mL of PBS. 3 mL of absolute ethanol was added to the cells with vortex for fixation. All the cells were stored at -20°C until analysis. On the day of analysis, cells were centrifuged (2000 rpm, 10 min), washed with PBS, suspended in 500 μL of PI solution (50 μg/mL propidium iodide, 100 μg/ mL RNase A and 0.05% Tritin X-100), and incubated at 37°C for 30 min. After incubation, 3 mL of PBS was added, and the cells were pelleted (1500 rpm, 5 min), suspended in PBS at a final cell density of 5 - 105 cells/mL and transferred to a Falcon 2054 tube (BD Bioscience, UK) for flow cytometry analysis. Analysis of 20,000 events was done on a CyAn ADP flow cytometer (Beckman Coulter, UK) using Summit 4.3 version (Dako Clolrado, Inc, USA) and ungated data was gathered within 1 h.

DNA footprinting

DNase I digestions were conducted in a total volume of 8 μL. The labeled DNA fragment (2 μL, 200 counts s-1) was incubated for 30 min at room temperature in 4 μL TN buffer (10 mM Tris Base and 10 mM NaCl, pH 7) containing the required drug concentration. Cleavage by DNase I was initiated by addition of 2 μL of DNase I solution (20 mM NaCl, 2 mM MgCl2, 2 mM MnCl2 and DNase I 0.02 Units, pH 8) and stopped after 3 min by snap freezing the samples on dry ice. The samples were subsequently lyophilized to dryness and resuspended in 5 μL of formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% cyanol blue). Following heat denaturation for 5 min at 90°C, the samples were loaded on a denaturing polyacrylamide (10%) gel containing urea (7.5 mM). Electrophoresis was carried out for 2 h at 1650 V (~70 W, 50°C) in 1- TBE buffer. The gel was then transferred onto Whatman 3MM and dried under vacuum at 80°C for 2 h. The gel was exposed overnight to Fuji medical X-ray film and developed on a Konica Medical Film Processor SRX-101A.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.