J Drug Target. 2007 Aug-Sep;15(7-8):475-86.

Fang J, Nakamura H, Iyer AK.

Laboratory of Microbiology and Oncology, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan. fangjun@ph.sojo-u.ac.jp

http://www.ncbi.nlm.nih.gov/pubmed/17671894

Abstract:

Reactive oxygen species (ROS), such as superoxide anion radicals (O.-2) and hydrogen peroxide (H2O2) are potentially harmful by-products of normal cellular metabolism that directly affect cellular functions. ROS is generated by all aerobic organisms and it seems to be indispensable for signal transduction pathways that regulate cell growth and reduction-oxidation (redox) status.

However, overproduction of these highly reactive oxygen metabolites can initiate lethal chain reactions, which involve oxidation and damage to structures that are crucial for cellular integrity and survival. In fact, many antitumor agents, such as vinblastine, cisplatin, mitomycin C, doxorubicin, camptothecin, inostamycin, neocarzinostatin and many others exhibit antitumor activity via ROS-dependent activation of apoptotic cell death, suggesting potential use of ROS as an antitumor principle.

Thus, a unique anti-cancer strategy named "oxidation therapy" has been developed by inducing cytotoxic oxystress for cancer treatment.

This goal could be achieved mainly by two methods, namely:

(i) inducing the generation of ROS directly to solid tumors and

(ii) inhibiting the antioxidative enzyme (defense) system of tumor cells.

Since 1950s, many strategies have been employed based on the first method, namely, administration of ROS per se (e.g. H2O2) or ROS generating enzyme to tumor bearing animals. However no successful and practical results were obtained probably because of the lack of tumor selective ROS delivery and hence resulting in subsequent induction of severe side effects.

To overcome these obstacles, we developed polyethylene glycol (PEG) conjugated O.-2 or H2O2-generating enzymes, xanthine oxidase (XO) and D-amino acid oxidase (DAO) (PEG-DAO) respectively. More recently, a pegylated (PEG) zinc protoporphyrin (PEG-ZnPP) and a highly water soluble micellar formulation of ZnPP based on amphiphilic styrene maleic acid (SMA) copolymer, SMA-ZnPP, are prepared, which are potent inhibitors of heme oxygenase-1 (HO-1).

HO-1 is a major antioxidative enzyme of tumors, that is different in mechanism of catalase or superoxide dismutase (SOD). Consequently, both PEG-enzymes and PEG-ZnPP exhibited superior in vivo pharmacokinetics than their parental molecules, particularly in tumor delivery by taking advantage of the EPR effect of macromolecular nature, and thus showed remarkable antitumor effects suggesting the potentials of this anticancer therapeutic for clinical application.

Furthermore, it has been well known that many antioxidative enzymes such as catalase, SOD are down-regulated in most solid tumors in vivo. On the contrary, HO-1 is highly upregulated and it plays a very important role of antioxidation, because HO-1 generates biliverdin, which being converted to bilirubin exhibits a very potent antioxidative effect, and hence antiapoptosis in tumors.

Thus this oxidation therapy, by inhibiting this HO-1 dependent antioxidant (bilirubin) formation by ZnPP, and by enhancing ROS generation, is expected to offer a powerful therapeutic modality for future anticancer therapy.

Mol Pharmacol. 2005 Oct;68(4):983-94. Epub 2005 Jul 15.

Lu HR, Zhu H, Huang M, Chen Y, Cai YJ, Miao ZH, Zhang JS, Ding J.

Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Peoples Republic of China.

http://www.ncbi.nlm.nih.gov/pubmed/16024664

Abstract:

Reactive oxygen species (ROS) are produced by all aerobic cells and have been implicated in the regulation of diverse cellular functions, including intracellular signaling, transcription activation, proliferation, and apoptosis. Salvicine, a novel diterpenoid quinone compound, demonstrates a broad spectrum of antitumor activities.

Although salvicine is known to trap the DNA-topoisomerase II (Topo II) complex and induce DNA double-strand breaks (DSBs), its precise antitumor mechanisms remain to be clarified. In this study, we investigated whether salvicine altered the levels of ROS in breast cancer MCF-7 cells and whether these ROS contributed to the observed antitumoral activity.

* Our data revealed that salvicine stimulated intracellular ROS production and subsequently elicited notable DSBs.

* The addition of N-acetyl cysteine (NAC), an antioxidant, effectively attenuated the salvicine-induced ROS enhancement and subsequent DNA DSBs.

* Heat treatment reversed the accumulation of DNA DSBs, and the addition of NAC attenuated the Topo II-DNA cleavable complexes formation and the growth inhibition of salvicine-treated JN394top2-4 yeast cells, collectively indicating that Topo II is a target of the salvicine-induced ROS.

* On the other hand, when examining the impact of salvicine on DNA repair pathways, we unexpectedly observed that salvicine selectively down-regulated the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) protein levels and repressed DNA-PK kinase activity; both of these effects were attenuated by NAC pretreatment of MCF-7 cells.

* Finally and most importantly, NAC attenuated salvicine-induced apoptosis and cytotoxicity in MCF-7 cells.

Conclusion:

These results indicate that apart from its direct actions, salvicine generates ROS that modulate DNA damage and repair, contributing to the comprehensive biological consequences of salvicine treatment, such as DNA DSBs, apoptosis, and cytotoxicity in tumor cells.

The finding of salvicine-induced ROS provides new evidence for the molecular mechanisms of this compound.

Moreover, the effects of salvicine-induced ROS on Topo II and DNA-PK give new insights into the diverse biological activities of ROS.

Nutr Cancer. 2010;62(5):601-10.

Reddivari L, Vanamala J, Safe SH, Miller JC Jr.

Colorado State University, Fort Collins, Colorado, USA.

http://www.ncbi.nlm.nih.gov/pubmed/20574921

Abstract:

We recently reported that colored potato extracts and an anthocyanin rich fraction suppressed lymph-node carcinoma of the prostate (LNCaP) and prostate cancer-3 (PC-3) prostate cancer cell proliferation and induced apoptosis via caspase-dependent and caspase-independent pathways.

* Chlorogenic acid, caffeic acid, gallic acid, catechin, malvidin, and glycoalkaloids (alpha-chaconine and solanine) have now been identified as the major bioactive components of potato, and their effects on LNCaP and PC-3 cell proliferation and apoptosis have been investigated. alpha-chaconine (5 microg/ml) and gallic acid (15 microg/ml) exhibited potent antiproliferative properties and increased cyclin-dependent kinase inhibitor p27 levels in both cell lines.

* Both alpha-chaconine and gallic acid induced poly [adenosine diphosphate (ADP)] ribose polymerase cleavage and caspase-dependent apoptosis in LNCaP cells; however, caspase-independent apoptosis through nuclear translocation of endonuclease G was observed in both LNCaP and PC-3 cells. alpha-chaconine and gallic acid activated c-Jun N-terminal protein kinase (JNK), and this response played a major role in induction of caspase-dependent apoptosis in LNCaP cells; whereas modulation of JNK and mitogen-activated protein kinase did not affect alpha-chaconine- and gallic acid-induced caspase-independent apoptosis.

* These results suggest that apoptosis induced by whole potato extracts in prostate cancer cell lines may be in part due to alpha-chaconine and gallic acid.

* L-arginine is a chemical building block called “an amino acid.” It is obtained from the diet and is necessary for the body to make proteins. L-arginine is found in red meat, poultry, fish, and dairy products. It can also be made in a laboratory and used as medicine.

* L-arginine is used for heart and blood vessel conditions including congestive heart failure (CHF), chest pain, high blood pressure, and coronary artery disease. L-arginine is also used for recurrent pain in the legs due to blocked arteries (intermittent claudication), decreased mental capacity in the elderly (senile dementia), erectile dysfunction (ED), and male infertility.

* Some people use L-arginine for preventing the common cold, improving kidney function after a kidney transplant, high blood pressure during pregnancy (pre-eclampsia), improving athletic performance, boosting the immune system, and preventing inflammation of the digestive tract in premature infants.

* L-arginine is used in combination with a number of over-the-counter and prescription medications for various conditions. For example, L-arginine is used along with ibuprofen for migraine headaches; with conventional chemotherapy drugs for treating breast cancer; with other amino acids for treating weight loss in people with AIDS; and with fish oil and other supplements for reducing infections, improving wound healing, and shortening recovery time after surgery.

* Some people apply L-arginine to the skin to speed wound healing and for increasing blood flow to cold hands and feet, especially in people with diabetes. It is also used as a cream for sexual problems in both men and women.

* L-arginine is converted in the body into a chemical called nitric oxide. Nitric oxide causes blood vessels to open wider for improved blood flow.

* L-arginine also stimulates the release of growth hormone, insulin, and other substances in the body.

* L-arginine increases blood flow and seems to decrease blood pressure.

* Sildenafil (Viagra) can lower blood pressure. L-arginine can also lower blood pressure. Taking sildenafil (Viagra) and L-arginine together might cause the blood pressure to go too low. Blood pressure that is too low can cause dizziness and other side effects.

Other Names:

2-Amino-5-guanidinopentanoic Acid, (2S)-2-Amino-5-{[amino (imino) methyl]amino}pentanoic Acid, Acide 2-Amino-5-guanidinopentanoïque, Arg, Arginine, Arginine Ethyl Ester, Arginine Ethyl Ester Dihydrochloride, Arginine Ethyl Ester HCl, Arginine HCl, Arginine Hydrochloride, Di-Arginine Malate, Di-Arginine Orotate, Di-L-Arginine-L-Malate, Dl-Arginine, L-Arginina, L-Arginine Ethyl Ester Dichloride, L-Arginine HCl, L-Arginine Hexanoate, L-Arginine Hydrochloride, L-Arginine Ketoisocaproic Acid, L-Arginine L-Pyroglutamate, L-Arginine Pyroglutamate, L-Arginine Taurinate, Malate de Di-Arginine, Orotate de Di-Arginine, R-Gene 10.

Asparagine, the beta-amido derivative of aspartic acid, is one of the 20 building blocks of protein.

* Asparagine is nonessential to the diet since the body can synthesize it. Only the l-stereoisomer participates in the biosynthesis of mammalian proteins. Its structure is identical to that of the amino acid aspartic acid, except that the latter compound's acidic side-chain carboxyl group has been coupled with ammonia, yielding an amide.

* Asparagine has carboxamide as the side chain's functional group.

* Asparagine is the ß-amide of aspartic acid synthesized from aspartic acid and ATP (adenosine triphosphate).

* The first amino acid to be isolated from its natural source, asparagine was purified from asparagus juice in 1806; proof of the occurrence of this amino acid in proteins was finally obtained in 1932.

* Asparagine plays an important role in the biosynthesis of glycoproteins and is also essential to the synthesis of a large number of other proteins.

* Asparagine is normally available as a white crystalline solid which dissolves in water.

* Asparagine can be produced within a cell through an enzyme called asparagine synthetase or it can absorbed into the cell from the outside.

* Asparagine is one of the principal and frequently the most abundant amino acids involved in the transport of nitrogen.

* Asparagine is an amino acid required by cells for the production of protein.

* Asparagine is an essential component of those proteins that are concerned with signalling, neuronal development and transmission across nerve endings.

* Asparagine is essential to all living cells for the production of many proteins.

* Cells can either internally produce asparagine or they can absorb asparagine from outside the cell, as it is obtained from a person's diet and made available through the bloodstream to all cells in the body.

* L-asparagine is an amino acid involved in the metabolic control of cell functions in nerve and brain tissue.

* Asparagine is very active in converting one amino acid into another (amination and transamination) when the need arises.

* Asparagine serves as an amino donor in liver transamination processes. In the liver, a function of asparagine involves converting one amino acid to another.

* Asparagine helps maintain an equilibrium of the central nervous system and has therapeutic properties, but is toxic when used in excess. It also participates in metabolic control of the brain and nervous system having some therapeutic uses in these areas.

* In the central nervous system, asparagine is needed to maintain a balance, preventing over nervousness or being overly calm. Like glutamine, asparagine is important in the metabolism of toxic ammonia in the body.

* The relatively unreactive, neutral amide group in the side chain of asparagine confers no special properties upon this amino acid once it is included within a protein by two peptide bonds.

* Both asparagine and glutamine are made with high-energy ATP and can return this energy when they metabolize back to aspartic acid and glutamic acid respectively. Both require vitamin B6 and enzymes for their formation.

* In plants, asparagine is a reversible combination of ammonia and aspartic acid. This is important in plant metabolism in order to preserve ammonia.

Asparagine Deficiency:

Asparagine deficiency is rare. However, an asparagine deficiency could be a contributing cause of fatigue and immune system stress including autoimmune disorders, infections and severe allergies.

1O2 Singlet Oxygen - PDT PhotoDynamic Therapy - Doctoral Thesis

Section 1.2.2: Mechanisms of Tumour Destruction

While it has long been known that the basis mode of cell death in PDT is mediated through singlet oxygen generation and other reactive oxygen species, and that direct cellular damage and vascular shutdown contribute to destruction of the tumour, only in recent years the importance of immune responses have been recognized [3].

Direct Cellular Damage: It has been shown that exposure of tumours to PDT in vivo can reduce the clonogenic tumour cells thought direct photodamage, i.e., those cells able to reproduce a tumour. However, complete tumour eradication is not always fully realized by this mechanism mainly due to two reasons: non-homogeneous distribution of the photosensitiser within the tumour and oxygen availability within the tissue that is targeted by PDT [4,6].

Vascular Damage: The viability of tumour cells also depends on the amount of nutrients supplied by the blood vessels, which in turn depend on growth factors produced by tumour or host cells. The mechanisms underlying the vascular effects of PDT differ greatly with different photosensitizers but vascular constriction, thrombus formation and inhibition of tumour growth have been identify among the most important ones [13].

Immune Responses: Several studies have reported that infiltration of lymphocytes, leukocytes and macrophages into PDT-treated tissue produced an activation of the immune response that consequently eliminates surviving tumour cells that have escaped to the direct effects of PDT [14-16].

PDT effects on all these targets may influence each other, producing a plethora of responses. However, the relative importance of each for the overall tumour response is yet to be defined. Further, it is known that both necrosis and apoptosis occur following PDT [17,18], albeit to different degrees depending on the photosensitiser and treatment conditions.

Necrosis or passive cell death can either result from extreme external physical conditions or severe cellular damage induced by chemical processes. It is usually accompanied by a loss of membrane integrity and associated with characteristic morphological changes such as organelle and cell swelling, bleb formation, loss of integrity of mitochondrial, lysosomal and plasma membranes and eventual breakdown
of the cell, leading to release of its contents into the surrounding area [19].

Apoptosis represents regulated cell suicide. It is a mechanism whereby organisms initiate cellular death via a process that is normally part of the genetic apparatus [20]. The end result is fragmentation of nuclear DNA and dissociation of the cell into membrane-bound particles that are phagocyted by neighbouring cells, preventing uncontrolled leakage of intracellular material to the environment and thereby avoiding
damage to neighbouring cells and tissue inflammation [21]. Apoptosis has been shown to be a rapid and dominant form of cell death following PDT in multiple experimental settings utilising various photosensitisers and cell types [22-24].

Louis Pasteur (1822-1895) and Antoine Béchamp (1816-1908), two French scientists, proposed diametrally opposed explanations on illness. Pasteur suggested thegerm theory – microbes produce illnesses. Béchamp offered thecellular theory – illnesses produce microbes. The first became famous, the second was forgotten. Who was right?

Béchamp (doctor, biologist, chemist, and physicist) discovered microbes before Pasteur. After years of observation, he was able to prove that human beings create their own microbes in order to re-establish order and balance inside tissues and organs. Béchamp also discovered microzymas, the smallest living particles. These changed their shape – pleomorphism – and became microbes when a person’s health deteriorated. Germs were actually allies. He published many scientific papers to prove his theory.

Pasteur (chemist) used and falcified Béchamp’s results, publishing them under his own name with opposite conclusions. Today, he is still considered one of humanity’s most cherished saviours – a good shepherd for the human flock (pasteur means “shepherd” in French). Numerous streets, organizations, and statues now bear his name. According to Pasteur, illnesses are due to aggressions from microscopic external pathogens. Each type of microbe has a specific shape and always cause the same disease. This theory is called monomorphism. To prevent illness, he proposed that we go to war against microbes. In fact, Pasteur was paid by the elite, who wanted to develop efficient eugenist methods.

Institut Pasteur and the entire field of vaccination were financed as early as 1887 by world banksters, the Rothschilds. They made sure that Pasteur’s theory became the norm across the planet. The illusive concept of the priority of the external world over the internal person sent its roots even deeper, keeping human beings the victims of invisible external enemies – microbes. Yet, Béchamp had proven the opposite. In fact, there is no external enemy. My body creates its own microorganisms in order to re-establish order and balance.

Several scientists later proved the existence of Béchamp’s microzymas: Royal Raymond Rife (polymorphic particles, 1920), Günther Enderlein (protits, 1925), Wilhelm Reich (bions, 1930), and Gaston Naessens (somatids, 1959). On his death bed, Pasteur admitted: “The terrain is everything, the germ is nothing.” Too late!Vaccination, the biggest crime against humanity, was alive and well. The elite had found their ultimate weapon.

Forbidden Treatments

Today, Pasteur’s germ theory replaces religion’s divine punishment. The mission of its new priests – scientists and doctors – consists of casting out microbes instead of demons. Yet, human beings remain the eternal victims of external enemies, an illusion that serves directly the interest of the elite in their attempt to control humanity. Any medical treatment that goes against their purpose is banished. Here a few examples of censured treatments. The first ten were compiled in North America by Daniel Haley. There are thousands more all over the world. Most of the researchers who developed them had to face accusations, legal proceedings, police arrests, threats to family members or themselves, and assassinations. This explains why 15 world-class microbiologists died in strange circumstances between September 11, 2001 and June 2, 2002.

Harry Hoxsey (USA/Mexico, 1901-1974) discovered the Hoxsey treatment, a natural tritherapy against cancer. After five prison sentences, he moved to Mexico. For the past 80 years, his treatment has enjoyed a success rate of 80%.

William Koch (US, 1885-1967) used glyoxylide to treat cancer, tuberculosis, polio, asthma, diabetes, leprosy, etc. He had to move to Brazil to continue treating patients.

Royal Raymond Rife (US, 1888-1971) built a live-cell microscope with a maximum magnification of 60,000 times and was able to prove Béchamp’s pleomorphism. He invented a ray tube that used specific vibrations to destroy each type of microbe. His laboratory and his research papers were destroyed.

Andrew Ivy (US, 1893-1978) developed a cancer-destroying serum called krebiozen, which he used successfully on thousands of patients. Prosecuted by the American Medical Association (AMA) and the Federal Drug Administration (FDA), he faced bankruptcy because of enormous legal expenses and stopped his research under threat of life imprisonment.

Robert Becker (US, 1923-2008) studied electromedicine and its use in cellular regeneration. His research funds were completely stopped when he publicly exposed the dangers of electropollution.

Stanley Jacob (US, 1924) popularized dimethyl sulphoxyde (DMSO), the chemical with widest range of therapeutic applications in the world. More than 3,000 studies on DMSO proved its efficiency for many types of illnesses. Its use is still blicked by the AMA and the FDA.

Herb Saunders (USA) injected microbes into pregnant cows and used their colustrum (first milk) to treat tuberculosis, cancer, arthritis, multiple sclerosis, etc. His instruments were confiscated and he had to face legal proceedings.

Gaston Naessens (France/Canada, 1924) invented the somatoscope in 1949, a live-cell microscope with a maximum magnification of 30,000 times. With this apparatus, he proved the pleomorphic capabilities of somatids – the fundamental and immortal particles of life. He invented a cancer remedy called 714X and was prosecuted in France for the illegal practice of medicine. He left for Corsica, then Quebec. The medical authorities made sure that his remedy remains unaccessible in Canada, except for terminally ill patients.

Hulda Clark (USA/Mexico, 1928-2009) established a link between illness and the presence of internal parasites, and invented an electric method for their destruction. She moved to Mexico after major legal proceedings.

Joseph Gold (USA) used hydrazine sulfate with 50% success rate for terminal cancer. The treatment is still banned in the US.

Stanislav Burzynski (Poland/US, 1943) discovered natural agents that eliminate cancer – antineoplastons. The FDA acquitted him after 25 years of persecution. As in Naessens’ case, his treatment is only available for terminally ill patients, under presctiption of a medical doctor.

Rick Simpson (Canada) popularized the use of hemp oil (cannabis), a remedy that has been in use for the past 5,000 years. A few decades ago, the elite had prohibited its use by declaring it an illegal drug. Simpson was prosecuted and the police vandalized his property.

This short North American list represents a tiny part of the treatments available across the planet that have been deliberately stopped by the elite in order to keep humanity living in fear, illness, and death. In fact, organizations such as the AMA and the FDA, and their equivalent in each country, have one single mission: stopping the propagation of efficient treatments.

That explains why we are actually witnessing the programmed disappearance of homeopathy, herbal medicine, gemmotherapy, traditional Chinese medicine, aromatherapy, vibrational remedies, oligotherapy, and more. For example, since April 1, 2001, the therapeutic use of practically all medicinal plants has become illegal in the European Union. The North American approach is slightly different, but its effect remains as devastating.

The European Directive on Traditional Herbal Medicinal Products (THMPD) has espablished strict rules for the use of such products in order to make themunavailable on the market. Even if a plant has been in use for thousands of years, companies or individuals selling them must now make sure that it has undergone the same type of testing as pharmaceutical drugs. The cost of such an operation varies between 90,000 and 140,000 euros. Also, each plant inside a mixture must be tested separately. This is more than most small manufacturers can afford. The medical Mafia has reached its goal. Mission accomplished!

Ukrain Therapy - (Click here)

Ukrain Therapy

1. Summary:The influence of intravenous injections of Ukrain (1mg/kg) on the oxygen tension (р02) in muscle, the oxygen saturation rate and oxygen utilization rate as well as on the respiration and oxidative phosphorylation indices of the liver mitochondria in mice with melanoma B-16 was studied by polarographic method.

Ukrain was shown to improve the oxygen delivery to the muscular tissue and to decrease a destructive effect of the malignant process on liver bioenergetics. The respiration rate of mitochondria during phosphorylation of exogenous ADP and the respiratory control coefficient were higher in the treated mice as compared to the untreated tumor-bearing animals. The drug significantly inhibited the growth of the primary tumor and its metastasis in lungs

2. Summary: The effects of L-arginine, the physiological precursor of nitric oxide (NO), and inhibitors of NO-synthase on the antinociceptive action of Ukrain (4.75, 9.5, and 19.0 mg/kg i.p.) were investigated using the writhing syndrome test in mice. It was found that L-arginine (0.1 or 1.0 mg/kg i. p.) significantly decreased or enhanced the antinociceptive effect of Ukrain, depending on the combination administered.

In addition, the inhibitors of NO-synthase NG-nitro-L-argίnine methyl ester (L-NAME) (1.0 and 10 mg/kg i. p.), 7-nitroindazole (1.0 mg/kg i. p.) and NG-monomethyl-L-arginine acetate (L-NMMA) (1.0 mg/kg i. p.) significantly enhanced Ukrain-induced antinociception. These results suggest that endogenous NO can modify the antinociceptive effect of Ukrain.

Ukrain Therapy - Greater Celandine Plant Extract:

Cytotoxic Activity of Proteins Isolated from Extracts of Coryalia cava Tubers in Human Carvical Carcinoma HeLa Cells

Conclusions:

Presented studies confirm that biological activity of C. cava extracts may also be related to proteins contained in the extracts. For possible further applications, the biologically active plant proteins should be separated from alkaloids and other secondary metabolites, which in higher doses might be toxic [14].

The studies represent preliminary demonstration of the effect exerted by tuber proteins from a plant of pharmacological significance on cells of a tumour cell line. In the subsequent studies, an effort will be made to isolate the individual proteins and examine their effect on tumour cells individually and in combinations.

Discussion:

Plants that belong to family Papaveraceae, like Greater Celandine (Chelidonium majus L.) and Corydalis cava Schweig, & Koerte, are a rich source of various biologically active substances with strong pharma cological activity. The mechanism of this activity is still unknown, but very important compounds are the proteins contained in the plants. Our earlier studies showed that biological activity of Chelidonium majus milky sap may be related to its protein content .

* The protein fractions which contained two nucleases induced apoptosis in human cervical cancer HeLa cells [17].

Most probably, the present study represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines. The cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein.

Results of protein identification in fractions from purification of C. cava tuber extracts using LC-ESI-MS/MS analysis have shown that proteins contained in bands of fractions 16-18 belong to plant pathogenesis - and defense-related proteins, indirectly or directly involved in plant defense reactions against stresses of various kind. Many of them belong to the PR protein family involved in plant defense against the pathogen attack. The family includes 17 protein classes of variable activities [24]. C. cava tubers are exposed to the attack of fungal, viral, bacterial and other pathogens during the entire vegetation season as well as during winter time [25].

* Accumulation of high number and variability of defense proteins in tubers together with several secondary metabolites, such as isoquinoline alkaloids, provides an effective and long-time protection against attack of pathogens.

*Moreover, defense-related proteins contained in C. cava tubers resemble 21 proteins identified earlier by our group in Chelidonium majus milky sap [15].

The studies represent preliminary demonstration of the effect exerted by tuber proteins from a plant of pharmacological significance on cells of a tumour cell line. The investigated protein fractions comprise a mixture of plant defense-related proteins. In subsequent studies, an effort
will be made to isolate the individual proteins and examine their effect on tumour cells individually and in combinations as well. Nevertheless, the synergistic action of all the compounds present in plant protein extracts is very important for their activity.

Many effective and extensively studied compounds of plant origin are, in fact, crude protein mixtures, such as phytotherapeutical drug bromelain, which is crude, aqueous extract from the stems and immature fruits of pineapples (Ananas comosus from the family Bromeliaceae), constituting a complex mixture of different thiol-endopeptidases and other components such as phosphatases, glucosidases, peroxidases, cellulases, glycoproteins, carbohydrates and several proteinase inhibitors [26].

Also, Viscum album L. extracts (VAE, European mistletoe) are composed of pharmacologically relevant compounds like: mistletoe lectins (ML I, II and III), viscotoxins and other low molecular proteins, VisalbCBA (Viscum album chitin-binding agglutinin), oligo- and polysaccharides, flavonoids, vesicles, triterpene acids, and others. Whole VAE as well as several of the compounds are cytotoxic and the mistletoe lectins have strong apoptosis-inducing effects [27].

Although the mechanism of presented activity of C. cava proteins is unknown, their defensive role for the plant could be very important. Many plant defense - related proteins belong to small (14-40 amino acids ), linear , cationic peptides.

*These peptides have membrane lytic properties and potent activity against a broad spectrum of microorganisms. They organize into ordered secondary structures (a-helix and b-sheet) in the membrane [28].

*Above a threshold concentration, peptides disturb the cell membrane and cause cell death due to membrane disintegration [29].