"Anti-Cancer Drugs 2000", 11, pp. 455-463

Report

AnvirzeI™, an extract of Nerium oleander, induces cell death in human but not murine [mouse] cancer cells

Sen Pathak,^1,2 Asha S Multani,¹ Satya Narayan,³ Virendra Kumar³ and Robert A Newman⁴ Departments of ¹ Cancer Biology and ² Laboratory Medicine, and ⁴ Pharmaceutical Development Center, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. ³ Shands Cancer Center and Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA.

Anvirzel™ (oleander extract) is a novel antitumor compound extracted from a flowering plant, N. oleander, belonging to the family Apocynaceae. Anvirzel™ consists of several compounds, including complex polysaccharides, proteins and individual sugars. It contains non-water soluble compounds, and two of these have been specifically identified by molecular weight and fragmentation characterization as Oleandrin and Oleandrigenin (Newman et al., unpublished observations). These two compounds of Anvirzel™, and possibly a third one, are cytotoxic. In the present studies, we examined the cytotoxicity of Anvirzel™ and Oleandrin on cancer cell lines of human, murine and canine origin. Our results from tests on cultures of two human prostate cancer cell lines, PC-3M and C4-2, and a murine melanoma cell line, K1735-X21, treated with Anvirzel™ and Oleandrin indicate several novel features: (i) neither Anvirzel™ nor Oleandrin showed any cytotoxic effect on murine melanoma K1735-X21 cells, (ii) when using even low drug concentrations human prostate cancer PC-3M cells showed significant susceptibility to cell killing, (iii) the cell killing is apparently mediated through the loss of telomeric DNA, followed by the arrest of cells in G2/M phase, induction of endomitosis, extensive DNA fragmentation, reduced levels of TRF2 and finally cell death, (iv) FACS analysis revealed induction of cell death in a dose and duration dependent manner in the human PC-3M cell line, followed by a saturation effect.

In earlier studies, Anvirzel™ was shown to have toxic effects on cells, decreasing the level of fibroblast growth factor-2. 15 Whether Anvirzel™ and its derivative Oleandrin exert their cytotoxic effects through inducing aberrations of chromosome morphology, polyploidy and cell death is not known. Because Anvirzel™ is known to have antitumor activity, we investigated the mechanism of cancer cell death in a variety of cancer cell lines of human, murine and canine origin. Our results show that both AnvirzelTM and Oleandrin are potent cell death inducers in two human prostate cancer cell lines.

Also interesting was the finding of a loss of telomere length and its correlation with TRF2 levels in the different cancer cell lines after their treatment with Anvirzel™.

A role for TRF2 has been implicated in telomere loss and end-to-end fusion of chromosomes leading to apoptosis; these results thus suggest that Anvirzel™-induced cell killing may be caused by the reduced levels of TRF2 in these cell lines and TRF2 reduction may be one mechanism of action whereby these drugs induce cancer cell apoptosis.

Conclusion

This study has shown that, in vitro, both Anvirzel™ and its derivative compound Oleandrin are highly cytotoxic to human prostate cancer cells.

Finally, Anvirzel™ and Oleandrin both have the potential to be used in chemotherapy for a variety of human cancer types. Studies evaluating such therapies are in progress at our Cancer Center.

"American Association for Cancer Research, Volume 60, Number 14, July 15, 2000", pp. 3807-3812 and 3838-3847 (2 Articles)

Report - Article 1

Cardiac Glycoside Stimulate CA²⁺ Increases and Apoptosis in Androgen-independent, Metastatic Human Prostate Adenocarcinoma Cells

David J. McConkey,¹ Yun Lin, Leta K. Nutt, Huseyin Z. Ozel, and Robert A. Newman Departments of Cancer Biology [D.J.M., L. K. N. J.] and Experimental Therapeutics [Y. L., R. A. N.]. University of Texas M.D. Anderson Cancer Center, Houston, TX 77030. and Ozelle Pharmaceuticals, Inc., San Antonio, Texas 78230 [H. Z. O.]

Abstract

Cardiac glycosides are used clinically to increase contractile force in patients with cardiac disorders. Their mechanism of action is well established and involves inhibition of the plasma membrane Na⁺ /K⁺ -ATPase, leading to alterations in intracellular K⁺ and Ca²⁺ levels. Here, we report that the cardiac glycosides oleandrin, ouabain, and digoxin induce apoptosis in androgen-independent human prostate cancer cell lines in vitro. Cell death was associated with early release of cytochrome c from mitochondria, followed by proteolytic processing of caspases 8 and 3. Oleandrin also promoted caspase activation, detected by cleavage poly (ADP-ribose) polymerase and hydrolysis of a peptide substrate (DEVD-pNA). Comparison of the rates of apoptosis in poorly metastatic PC3 M-Pr04 and highly metastatic PC3 M-LN4 subclones demonstrated that cell death was delayed in the latter because of a delay in mitochondrial cytochrome c release. Single-cell imaging of intracellular Ca²⁺ fluxes demonstrated that the proapoptotic effects of the cardiac glycosides were linked to their abilities to induce sustained Ca²⁺ increases in the cells. Our results define a novel activity for cardiac glycosides that could prove relevant to the treatment of metastatic prostate cancer.

Report - Article 2

Oleandrin Suppresses Activation of nuclear Transcription Factor-kB, Activator Protein-1 and c-Jun NH₂-Terminal Kinase¹

Sunil K. Manna, Nand K. Sah,² Robert A. Newman, Angela Cisneros, and Bharat B. Aggarwal³ Cytokine Research laboratory, Department of Bioimmunotherapy [S. K. M., N. K. S., B.B.A.], and Pharmaceutical Development Center [R. A. N., C. J.], University of Texas M.D. Anderson Cancer Center, Houston, TX 77030

Abstract

Agents that can suppress the activation of nuclear factor-kB (NF-kB) and activator protein-l (AP-l) may be able to block tumorigenesis and inflammation. Oleandrin, a polyphenolic cardiac glycoside derived from the leaves of Nerium oleander, is a candidate NF-kB and AP-l modulator. We investigated the effect of oleandrin on NF-kB activation induced by inflammatory agents. Oleandrin blocked tumor necrosis factor (TNF)-induced activation of NF-kB in a concentration- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of IkB α, an inhibitor of NF-kB. A proprietary hot water extract of oleander (Anvirzel^TM) also blocked TNF-induced NF-kB activation; subsequent fractionation of the extract revealed that this activity was attributable to oleandrin. The effects of oleandrin were not cell type specific, because it blocked TNF-induced NF-kB activation in a variety of cells. NF-kB-dependent reporter gene transcription activated by TNF was also suppressed by oleandrin. The TNF -induced NF-kB activation cascade involving TNF receptor l/TNF receptor-associated death domain/TNF receptor-associated factor 2/NF-kB-inducing kinase/IkB α kinase was interrupted at the TNF receptor-associated factor 2 and NF-kB-inducing kinase sites by oleandrin, thus suppressing NF-kB reporter gene expression. Oleandrin blocked NF-kB activation induced by phorbol ester and lipopolysaccharide. Oleandrin also blocked AP-l activation induced by TNF and other agents and inhibited the TNF-induced activation of c-Jun NH₂-terminal kinase. Overall, our results indicate that oleandrin inhibits activation of NF-kB and AP-l and their associated kinases. This may provide a molecular basis for the ability of oleandrin to suppress inflammation and perhaps tumorigenesis.

"Journal of Herbal Pharmacotherapy, Volume 1, Number 3, 2001", pp. 1-17

Report

Composition and Preliminary Pharmacology Studies with Anvirzel^TM: An Extract of Nerium oleander

Robert A. Newman, PhD, Angela Cisneros, MS, Edward Felix, MS, Mary Vijjeswarapu, BS, Yun Lin, BS, Peiying Yang, PhD, Parastoo Azadi, PhD Pharmaceutical Development Center, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030

Abstract

Initial identification and characterization of the major biochemical and biological properties of Anvirzel^TM, a patented hot-water extract of Nerium oleander, were undertaken using HPLC, LC/MS, and in vitro cell growth inhibition assays. Analyses using high pH anion exchange chromatography with electrochemical detection consistently revealed seven major polysaccharide peaks which were subsequently designated as a "carbohydrate fingerprint." Analyses of monomer sugar composition revealed glucose and galacturonic acid as major carbohydrate residues, while carbohydrate linkage studies identified glucopyranosyl and 6-glucopyranosyl as major terminal carbohydrate residues. Nonpolar compounds were separated from polar components through solid phase extraction and analyzed by both reverse phase HPLC and LC/MS methods. Two nonpolar cytotoxic components, oleandrin and its aglycone, oleandrigenin, were detected. Quantitative analysis showed that the oleander extract contained oleandrin and oleandrigenin at concentrations of 2.5 and 4.4 μ g/mg extract, respectively. Five proteins with molecular weights of 6, 20, 35, 68, and 150 kD were also identified in the oleander extract although their functions remain unknown. Cytotoxicity studies showed oleandrin to be a potent growth inhibitory compound against human melanoma BRO cells with an IC₅₀ of 4.0 ng/ml. In the same test system, the IC₅₀ values for oleandrigenin and the complete oleander extract against human melanoma cells were 17.0 ng/ml and 1.6 (μg/ml, respectively. This initial characterization and pharmacology research has served as a basis for quality control studies for the production and subsequent clinical Phase I trial of Anvirzel^TM .

"Biochemical Pharmacology 62(4), (2001)", pp. 469-472

Report

Inhibition of export of fibroblast growth factor-2 (FGF-2) from the prostate cancer cell lines PC3 and DU145 by Anvirzel and its cardiac glycoside component, oleandrin

Judith A. Smith^a, Timothy Madden^b,c, Mary Vijjeswarapu^b, Robert A. Newman^b,c,*, ^a Division of Pharmacy, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA, ^b Pharmaceutical Development Center, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA, ^c Department of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Box 52, 1515 Holcombe Blvd., Houston, TX 77030, USA

Abstract

Anvirzel^TM is an extract of Nerium oleander currently undergoing Phase I clinical evaluation as a potential treatment for cancer. Two of the active components of Anvirzel^TM are the cardiac glycosides oleandrin and oleandrigenin. Previous studies have demonstrated that, in vitro, cardiac glycosides may inhibit fibroblast growth factor-2 (FGF-2) export through membrane interaction with the Na⁺,K⁺-ATPase pump. In continuing research on the antitumor activity of this novel plant extract, the relative abilities of oleandrin and oleandrigenin to inhibit FGF-2 export from two human prostate cancer cell lines, DUl45 and PC3, were examined. An ELISA assay was utilized to determine the FGF-2 concentration in the cell culture medium before and after exposure to cardiac glycosides or the parent extract material Anvirzel^TM . Both cell lines were exposed to non-cytotoxic concentrations of oleandrin (0.05 and 0.1 ng/mL) for up to 72 hr. Studies also were conducted with Anvirzel^TM and ouabain. Oleandrin (0.1 ng/mL) produced a 45.7% inhibition of FGF-2 release from PC3 cells and a 49.9% inhibition from DU145 cells. Non-cytotoxic concentrations (100 ng/mL) of Anvirzel^TM produced a 51.9 and 30.8% inhibition of FGF-2 release, respectively, in the two cell lines. The decrease in FGF-2 release from cells required continuous incubation for 48-72 hr; shorter incubation times were not effective. These results demonstrate that Anvirzel^TM , like oleandrin, inhibited FGF-2 export in vitro from PC3 and DUI45 prostate cancer cells in a concentration- and time-dependent fashion and may, therefore, contribute to the antitumor activity of this novel treatment for cancer.

"Cancer Letters 185, (2002)", pp. 145-151

Report

Enhancement of radiotherapy by oleandrin is a caspase-3 dependent process

Sachiko Nasu^a, Luka Milas^a, Shinichiro Kawabe^a, Uma Raju^a, Robert A. Newman^b,*, ^a Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009, USA, ^b Pharmaceutical Development Center, University of Texas M.D. Anderson Cancer Center, 8000 El Rio, Houston, TX 77054, USA

Abstract

Cardiac glycosides such as digitoxin and ouabain have previously been shown to be selectively cytotoxic to tumor as opposed to normal cells. Moreover, this class of agents has also been shown to act as potent radiosensitizers. In the present study we explored the relative radiosensitization potential of oleandrin, a cardiac glycoside contained within the plant extract known as Anvirzel^TM that recently underwent a Phase I trial as a novel drug for anticancer therapy. The data show that oleandrin produces an enhancement of sensitivity of PC-3 human prostate cells to radiation; at a cell survival of 0.1, the enhancement factor was 1.32. The magnitude of radiosensitization depended on duration of exposure of cells to drug prior to radiation treatment. While a radiosensitizing effect of oleandrin was evident with only 1 h of cell exposure to drug, the effect greatly increased with 24 h oleandrin pretreatment. Susceptibility of PC-3 cells to oleandrin and radiation-induced apoptosis was dependent on activation of caspase-3. Activation was greatest when cells were exposed simultaneously to oleandrin and radiation. Inhibition of caspase¬3 activation with Z-DEVD-FMK abrogated the oleandrin-induced enhancement of radiation response suggesting that both oleandrin and radiation share a caspase-3 dependent mechanism of apoptosis in the PC-3 cell line.

"Journal of Experimental Therapeutics and Oncology 2, (2002)", pp. 1-8

Report

Murine pharmacokinetics and metabolism of oleandrin, a cytotoxic component of Nerium oleander

Dan Ni, Timothy L. Madden, Mary Johansen, Edward Felix, Dah H. Ho, and Robert A. Newman, Pharmaceutical Development Center, University of Texas M.D. Anderson Cancer Center, Houston, TX.

Abstract

Pharmacokinetic studies of [³H]oleandrin, a cardiac glycoside component of Anvirzel^TM, were conducted in mice after either an i.v. dose (40 μ g/kg) or a p.o. dose (80 μ g/kg). Oleandrin was rapidly absorbed after oral dosing (C_max at 20 min) although the elimination half life was longer (2.3 ± 0.5 h) than that after i.v. dosing (0.4 ± 0.1 h). The AUC_0-co values obtained after i.v. and p.o. dosing were 24.6 ± 11.1 and 14.4 ± 4.3 [(ng/ml)h], respectively, resulting in an oral bioavailability of approximately 30%. After i.v. administration, oleandrin concentration in liver was approximately twice that measured in heart or kidney tissue. Oleandrigenin, the aglycone of oleandrin, was also found in these tissues. At 5 min, >60% of the total radioactivity in liver was due to oleandrin while 28% of the given dose was present as oleandrigenin. Twenty-four hours following injection, 8% of total radioactivity was excreted in urine and contained both oleandrigenin (4.4% of the injected dose) and oleandrin (1.9%). Sixty-six percent of injected radioactivity was found in feces and consisted of oleandrin and oleandrigenin in equal amounts. Uptake of oleandrin in brain after i.p. injection of oleandrin (3 mg/kg) or oleander extract (700 mg/kg) was examined. Measured by LC/MS/MS, oleandrin content in brain was higher following injection of extract than it was with an equivalent dose of oleandrin. The data suggest that components within oleander extract may enhance transport of oleandrin across the blood brain barrier.

Want more?

1. Taylor A, McKenna GF, Burlage HM. Anticancer activity of plant extracts. Texas Reports on Biology and Medicine 1956;14:538-556.
2. Tarkowska JA. Effect of water extract from leaves of Nerium Oleander L. on mitosis. Acta Societatis Botanicorum Poloniae 1971;XL(4):623-631.
3. Tarkowska JA. Antimitotic action of glycosides of Nerium Oleander L. Hereditas 1971;67:205-210.
4. Statz D, Coon FB. Preparation of plant extracts for anti-rumor screen. Cancer Treatment Reports 1976;60:999-1005.
5. Hartwell JL. Types of anticancer agents isolated from plants. Cancer Treatment Reports 1976;60:1031-1067.
6. Duke J, at all. Medicinal plants of China. Reference Publication, 1985:97-98.
7. Copy of NCI SCREENING DATA SUMMARY ON NSC 251673.
8. The Merck Index, 10th Ed., pub. Merck&Co., Inc., Rahway, N.J., 1983:p.355,979,1413.
9. Woo WS, Lee EB, Han BH. Biological evaluation of Korean medicinal plants. Archives of Pharmaceutical Research 1979;2(2):127-131.
10. Chavan SR, Nikam ST. Studies on the larvicidal properties of Nerium indicum Mill (apocynaceae) leaves. Bulletin of the Hattdine Institute 1983;11(3):68-70.
11. Mansuri SM, Girdhar A, Doctor RB. Preliminary study of beta-adrenergic blocking action of Nerium odorum Linn. Indian Journal of Physiology & Allied Sciences 1980:34(I):30-31.
12. Karawya MS, Baldaa SI, Khayyal SE. Isolation of oleandrin and adynerin from Nerium oleander L. growing in Egypt. Egyptian Journal of Pharmaceutical Science 1973:14(2):113-116.
13. Leporatti ML, Posocco E, Pavesi A. Some new therapertic uses of several medicinal plants in the province of Terni. Journal of Ethnopharmacology 1985;14:65-68.
14. Yamauchi T, Abe F, Tachibana Y, Atal CK, Sharma BM, Imre Z. Quantitative variations in the cardiac glycosides of oleander. Phytochemistry 1983;22(10):2211-2214.
15. Szabuniewicz M, McCrady JD, Camp BJ. Treatment of experimentally induced oleander poisoning. Archives of Int Pharmacodynamics 1971;189:12-21.
16. Haynes BE, Bessen HA, Wightman WD. Oleander tea: herbal draught of death. Annals of Emergency Medicine 1985;14(4):350-353.
17. Schwartz WL, Bay WW, Dollahite JW, Storts RW, Russel LH. Toxicity of Nerium oleander in the monkey (cebus apella). Veterinary Pathology 1974;11:259-277.
18. Woo SW, Shin KH, Kwon YM. Biological evaluation of Korean medicinal plants. Journal of the Pharmaceutical Society of Korea 1972;16:121-128.
19. Elllis Md. Dangerous Plants, Snakes, Anthropods@Marine Life: Toxicity & Treatment. Hamilton, I11, Drug Intelligence Publ. Inc., 1978:32-33.
20. Osterloh J, Herold S, Pond S. Oleander interference in digoxin radioimmunoassay in a fatal ingestion. JAMA 1982;247:1596-1597.
21. Shaw D, Pearn J. Oleander Poisoning. Med J Aust 1979;2:267-269.
22. Mahin L, Marzou A, Huart A. A case report of Nerium Oleander posoning in cattle. Vet Hum Toxical 1984;26(4):303-304.
23. Karawya MS, Balbaa SI, Khayyal SE. Estimation of cardenolides in Nerium oleander. Planta Med 1973;23:70-73.
24. Ozel HZ. Kanser tedavisinde klinik vakalar. 4. Balkan tip gunleri. 16-20 Eylul 1973, Ankara.(Clinical cases in the treatment of cancer. 4th Balkanic medical days. 16-20 September 1973, Ankara.)
25. Ozel HZ. Kanser tedavisinde bir deneme (An essay in the treatment of cancer). Dirim 1974;4:172-176.
26. Ozel HZ. Zakkum usaresinin malign tumorlere etkisi uzerine klinik calismalar (Clinical studies about the effect of Nerium oleander extract on malignant tumors). Dirim 1974;12:565-572.
27. Report on the evaluation tests of NOI performed at Sandoz pharmaceutical company's Basel and Vienna laboratories in 1987.
28. Carbik I, Baser KHC, Ozel HZ, Ergun B, Wagner H. Immunologically Active Polysaccharides from the Aqueous Extract of Nerium Oleander. Planta Med 1990;56:668 .
29. Ozel HZ, Baser KHC, Carbik I, Wagner H, inventors; Polysaccharide mixture with immune stimulating and anti-proliferating effect, method of production and medicines containing the substances. Canadian patent application
    CA 2016948 filed on May 16, 1990.
30. Ozel HZ, inventor. Extracts of Nerium species, methods of preparation, and use therefore. US patent 5,135,745. 1992 Aug 4.
31. McConkey DJ, Lin Y, Nutt LK, Ozel HZ, Newman RA. Cardiac glycosides stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic human prostate adenocarcinoma cells. Cancer Res 2000 Jul 15;60(14):3807-12.
32. Wang X, Plomley JB, Newman RA, Cisneros A. LC/MS/MS analyses of an oleander extract for cancer treatment. Anal Chem 2000 Aug 1;72(15):3547-52.
33. Pathak S, Multani AS, Narayan S, Kumar V, Newman RA. Anvirzel, an extract of Nerium oleander, induces cell death in human but not murine cancer cells. Anticancer Drugs 2000 Jul;11(6):455-63.
34. Smith JA, Madden T, Vijjeswarapu M, Newman RA. Inhibition of export of fibroblast growth factor-2 (FGF-2) from the prostate cancer cell lines PC3 and DU145 by Anvirzel and its cardiac glycoside component, oleandrin. Biochem Pharmacol 2001 Aug 15;62(4):469-72.
35. Ni D, Madden TL, Johansen M, Felix E, Ho DH, Newman RA. Murine pharmacokinetics and metabolism of oleandrin, a cytotoxic component of Nerium oleander. J Exp Ther Oncol 2002 Sep-Oct;2(5):278-85.
36. Mekhail T, Kellackey C, Hutson T, Olencki T, Budd GT, Peereboom D, Dreicer R, Elson P, Ganapathi R, Bukowski R. Phase I study of Anvirzel in patients with advanced solid tumors. Proc Am Soc Clin Oncol 20:82b, 2001 (abstr 2077).
37. Duke J. Phytochemical and Ethnobotanical Databases , U.S. Department of Agriculture, Agricultural Research Service .
38. Ghoneum M, Ozel HZ, Gollapudi S. Nerium oleander leaf extract sensitizes human burkett cell lymphoma (Raji) to human cytotoxicity mediated by natural killer cells. Clinical Immunology, 2006, vol. 119, no. SUPP, pp. S188 .

I think that should be enough to make simple excreting functions rather painful for you for the near future, but while you are squirming, here is another tidbit for you to tuck in the area closest to your brain:  Oleander extract, the exact same oleander extract I write about and recommend, has passed FDA phase I trials.  Do you know what phase I trials are for?  TOXICITY.  And do you know what they found?  No unsafe toxic level was ever reached.  Instead the dosage reached a point beyond which it was impractical to increase.

And here is the final irony for you:  If a major pharmaceutical firm were to pick up the rest of the hundreds of millions it takes to complete the FDA trials, oleander would be an official approved drug and you would be promoting it to the high heavens.

Now, having failed miserably, let's return to the original proposition you dodged:  Once again, the task is simple:  find me one person who took properly made uncontaminated colloidal silver and turned blue or was harmed in any way.  Just one.  Find me one person who took my advice or who took oleander and was harmed in any way.  Just one.  You can't do it and so you and your mentor troll resort to slurs and lies and cut and paste mainstream justifications.

Mainstream medicine sold their souls for profit a long time ago.  Not sure when you and C sold yours, but the transaction is apparent and you continue to reveal yourself with each additional pathetic post you make here.

DQ (aka Tony Isaacs)