Selective Cytotoxic Diterpenes From Euphorbia Poissonii Biology Essay

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Cancer is one of the most dangerous, severe and high mortality diseases of XXI century all over the world. The worst situation takes place in the developing countries due to poverty and lack of quality drugs. Therefore, most of the peoples rely over folk/indigenous medicines derived from plants, etc. Plants of Euphorbiaceae have been used for remedy against cancer since ancient times. A variety of antibacterial, anti-viral, molluscicidal and tumor-promoting to tumor-inhibiting compounds have been isolated from these plants (I.B. Baloch et al./ European Journal of Medicinal Chemistry 43 (2008) 274-281)

Euphorbia family includes about 2000 species and has a wide distribution, with at least 750 species occurring in continental Africa and 150 species in Madagascar and the Indian Ocean Islands. Euphorbia poissonii belongs to subgenus section Euphorbia, large group which is characterised by succulent, angular stems, stipules modified into small spines, a spine shield with a pair of spines (sometimes fused into a single spine), axillary inflorescencens and seeds without caruncle.

The latex of Euphorbia poissonii is very caustic and toxic, and very irritating to the skin and mucous membranes. It can cause blindness when in contact with the eyes. Despite its toxicity, it is used medicinally. In Nigeria a few drops of latex are applied to Guinea-worm sores and to skin papilloma. A few drops of latex with sugar cane or in palm wine or soup are taken as a purgative.

The latex of Euphorbia poissonii contains esters of the diterpene tigliane type alcohols 12-deoxyphorbol and 12-deoxy-16-hydroxyphorbol, the diterpene daphnane type alcohol resiniferol, and several esters of the macrocyclic diterpene alcohol 19-hydroxyingol, e.g. the pentacyclic euphorianin. Most compounds isolated are mono- and di-esters of 12-deoxyphorbol. Daphnane esters are generally known for their potent skin irritant properties, whereas tigliane esters are toxic and tumour promoters; however, some of the tigliane compounds, especially 12-deoxyphorbol derivatives, possess anticancer activities.

The variety of irritant compounds present in high concentrations is reflected by the irritant activity of the latex, which is more than 30 times stronger in Euphorbia poissonii latex (ID50 = 0.1 μg / 5 μl) than in the latex of e.g. Euphorbia unispina, although after 24 hours both activities have almost the same value. The isolated aromatic esters of the daphnane type are more potent irritants in mouse ear tests than the aromatic tigliane esters, especially resiniferatoxin (ID50 = 0.00021 nMol / 5 μg) and tinyatoxin (ID50 = 0.0012 nMol / 5 μg). Of the 6 isolated aromatic tigliane esters, the highly irritant 12-deoxyphorbol-13-O-phenylacetate-20-O-acetate (ID50 = 0.075 nMol / 5 μg) is the major compound; candletoxin A, candletoxin B and DPP ( 12-deoxyphorbol 13-phenylacetate) are also strongly irritant. The irritant activity of resiniferatoxin and tinyatoxin is rapid. It reaches a maximum within 4 hours and then fades to inactivity after 24 hours.

Resiniferatoxin and tinyatoxin are highly toxic, as they bind to pain receptors in the same way as capsaicin, but much more powerfully. They stimulate the neurons to fire repeatedly until the neuron dies, causing searing pain and sending the victim into severe anaphylactic shock. Resiniferatoxin is used in the treatment of incontinence associated with an overactive bladder. It also has antifeedant and analgesic properties. Efforts have been made to synthesize this compound. In laboratory tests, DPP induced the expression of HIV-1 in latently infected T cells and rendered them sensitive to killing by an immunotoxin. DPP is 20- to 40-fold more potent than the related phorbol ester prostratin, and the combination of high potency and antitumor promoting activity makes DPP an attractive candidate for therapy of HIV-1 infection.

During further phytochemical studies on the latex, derivatives of 19-hydroxyingol were isolated. These compounds showed cytotoxic activities against 6 human solid tumour cell lines (lung carcinoma, breast carcinoma, colon adrenocarcinoma, kidney carcinoma, prostate adrenocarcinoma and pancreatic carcinoma). Moreover, most of the 12-deoxyphorbol ester derivatives showed selective cytotoxicity for the human kidney carcinoma cell line with potencies for one compound exceeding those of the anticancer drug adriamycin by 10,000 times.

30 years ago the hypothetical concept of cocarcinogens has been sustained by chemical, biochemical, and biological investigations of the polyfunctional, diterpene mono- and diester type promoters (and stimulators) of initiation, originated from various plant species. It was suspected that cocarcinogens of the diterpene ester type may play a role as possible carcinogenic risk factors of the human environment, especially as environmental promoters. Diterpenes from Euphorbiaceae and Thymelaeaceae were identified as highly active irritants and as cocarcinogens of the promoter type in mouse skin. In the given work research team leaded by Majekodunmi O. Fatope investigated cytotoxic activity of diterpenes extracted from latex of Euphorbia poissonii. Cytotoxic drugs are cellular poisons with very little cell selectivity. Their antitumor effect is usually based on cellular kinetics. In contrast to normal cells, cancer cells tend to divide continuously and are more readily exposed to the effects of cytotoxic agents. Therefore, the most common side effect that are clinically observed after chemotherapy are linked to body parts that divide continuously, such as marrow of intestinal mucosa. Most cytotoxic drugs directly affect the cell cycle during DNA replication or mitosis.

Figure 1. Mechanism of action of cytotoxic agents.

The 10% aqueous MeOH soluble fraction, of the latex extracts of this plant, exhibited a modest activity against the brine shrimp larvae (LC50 114 µg/mL). It is also showed a significant selectivity for certain cells when tested for cytotoxicity in a panel of human solid tumor cell lines in culture. Bioactivity-directed solid chromatographic fractionation of the bioactive fraction gave 12-deoxyphorbol 20-acetate 13-angelate (1), 12-deoxyphorbol 20-acetate 13-phenylacetate (2), 12-deoxyphorbol 13(9,10-methylene)undecanoate (3), 20-hydroxy-12-deoxyphorbal angelate (4), resineferol 20-(4-hydroxy-3-methoxyphenylacetate) 9,13,14-orthophenylacetate (5), and 20-hydroxyresineferol 9,13,14-orthophenylacetate (6).

3

2

1

6

5

4

Figure 2. Active compounds extracted from latex of Euphorbia poissonii

All compounds were undertaken in vitro and in vivo assay. For structure analysis high-resolution mass spectroscopy (HRFABMS), UV, NMR and IR spectrum analysis were used. Thus suggested a molecular formula C32H48O6 for the compound 3. This compound has a UV maximum at 235 nm, indicating the presence of an α,β-unsaturated carbonyl group. The C-NMR data of 3 displayed a tigliane skeleton, similar to 1, 2 and 4 together with 12-carbon substituent at C-13. The C-NMR spectrum also showed two trisubstituted double bonds, one keton and one ester carbon. The molecular formula of 3 suggested the presence of a total of nine unsaturation equivalents.

The structure of the substituent at C-13 of 3 was determined by a careful analysis of the DEPT and H-NMR signals of the 12-carbon ester moiety. The presence of one methyl group, one ester carbonyl carbon, two methines, and eight methylene carbons, one of which resonated at δ established an unusual cyclopropane undecanoate structure for the 12-carbon substituent.

The NMR spectra of compound 5 and 6 showed the characteristic features of a daphnane ortho ester diterpene. The identity of compound 5 was confirmed as resineferol 20-(4-hydroxy-3-methoxyphenylacetate) 9,13,14-orthophenylacetate from the spectral data. The NMR spectra of compounds 5 and 6 were quite similar except for the absence of the benzoate signals in compound 5.

Afterwards in vivo experiments took place.

The shrimp lethality assay was proposed by Michael et al. and later developed by Vanhaecke et al/ and Sleet and Brenel. It is based on the ability to kill laboratory-cultured Artemia nauplii brine shrimp. The brine shrimp cytotoxicity assay was considered as a convenient probe for preliminary assessment of toxicity, detection of fungal toxins, heavy metals, pesticides and cytotoxicity testing of dental materials. It can also be extrapolated for cell-line toxicity and anti tumor activity. Artemia salina belongs to the phylum Arthropoda, class Crustacea. Their life cycle begins by hatching of dormant cysts where these cysts are inactive but, once in salt water, they become rehydrated and resume their development. These larvae is characterized by common features such as adaptability to wide ranges of salinity (5-250g·L-1) and temperature (6-35 °C), short life cycle, high adaptability to adverse environmental conditions, high fecundity, bisexual/parthenogenetic reproduction strategy (with nauplii or cysts production), small body size, adaptability to varied nutrient resources as it is a non-selective filter feeder, sensitive to toxic substances and simple equipment used for the measurements. Dried cysts are performed and incubated in a hatcher with strong aeration under a continuous light regime. Approximately 12 h after hatching the phototropic nauplii are collected with a pipette from the lighted side and concentrated in a small vial. Ten brine shrimp are transferred to each well using adequate pipettes. Each test consists of exposing groups of 10 Artemia aged 12 h to various concentration of the toxic compound. The toxicity determinates after 12 h, 24 h and 48 h of exposure. The numbers of survivors are counted and percentage of deaths is calculated. Larvae are considered dead if they did not exhibit any internal or external movement during several seconds of observations. The larvae do not receive food. To ensure that the mortality observed in the bioassay could be attributed to bioactive compounds and not to food starvation, the dead larvae are compared in each treatment to the dead larvae in the control. In any case, hatched brine shrimp nauplii can survive for up to 48 h without food because they still feed on their yolk-sac. However, in cases where control deaths are detected, the percentage of mortality (%M) is calculated as: % M = percentage of survival in the control - percentage of survival in the treatment.

In the middle of XXth century strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. In the end of 1980s a new anticancer drug screening program based upon the use of multiple panels of human solid tumor cell lines is under development by the U. S. National Cancer Institute's Developmental Therapeutics Program, Division of Cancer Treatment. The goal of the new program was to evaluate experimental agents against groups of cell line panels each representing a major clinical category of human malignancy. Each panel (e.g., lung, colon, melanoma, renal, ovarian, and central nervous system) was to contain multiple, representative human tumor cell lines.

In the given work certain fractions and pure compounds were tested for cytotoxicity in a panel of ix human solid tumor cell lines, using a standard protocols for A-549 (lung carcinoma), MCF-7 (breast carcinoma), HT-29 (colon adrenocarcinoma), A-498 (kidney carcinoma), PC-3 (prostate adrenocarcinoma), and PACA-2 (pancreatic carcinoma) with adriamycin as appositive control

Results of the BST and of the cytotoxic activities against six human solid tumor ce3ll lines for compounds 1-6 are summarized in the Table 1. Conpound 3 is more than a thousand times as active as the others in the BST. Compounds 1-3, 5, and 6 showed a strong cytotoxic selectivity for the human kidney carcinoma (A-498) cell line.

Table 1. Lethality and Cytotoxicity of compounds 1-6 in the B ST Assay and Human Solid Tumor Cell Culture Systems

compound

LC50(µg/mL)

ED50(µg/mL)

BSTa

(A-549)b

(MCF-7)c

(HT-29)d

(A-498)e

(PC-3)f

PACA-2g

1

191,04

18,89

41,76

30,77

2,18*10-4

27,71

30,34

2

12,03

26,06

41,26

29,47

1,59*10-7

35,10

26,90

3

1,10*10-2

2,85*10-1

2,30

3,83*10-1

<10-7

2,44*10-1

1,92

4

114,65

11,60

69,88

26,50

45,96

17,93

37,25

5

280,79

1,12

4,29

3,00

1,08*10-1

2,90

2,44

6

n/t

5,41*10-2

30,88

7,41

1,97*10-2

4,56

3,55

adriamycin

n/t i

4,16*10-3

3,76*10-1

3,90*10-2

1,87*10-3

6,37*10-2

1,63*10-2

a Brine shrimp lethality test. b Lung carcinoma. c Breast carcinoma. d Colon adrenocarcinoma. e Kidney carcinoma. f Prostate adrenocarcinoma. g Pancreatic carcinoma. h Positive control. i n/t: not tested

Compound 5 was significantly less active but was still selective for kidney carcinoma. Of all the tigliane esters isolated, compound 3, hitherto unknown, demonstrated the strongest bioactivities and selectivity of other one million times for the human kidney carcinoma (A-498). This can be the result of the presence of the cyclopropyl ring in the fatty acid ester substituent at C-13 of compound 3. Compounds that show selective activity at ED50 values of 4 µg/mL and less in the cytotoxicity assay may be considered promising in our search for potential antitumor compounds from plant sources. Potent skin irritancy was noted when handling compound 5.

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