At the 89th annual meeting of the American Association for Cancer Research in New Orleans, March 28 – April 1, 1998 A. Panzer and J.C. Seegers (University of Pretoria, South Africa) presented their pioneering work on the selective effect of Ukrain on various cancer cell lines. Moreover, it has been first time as the fine mechanism of action of Ukrain had been described. The authors concluded in their paper “that Ukrain is selectively toxic to malignant cells by causing a metaphase block which is characterised by abnormal chromosomal distribution, and results in the formation of micronuclei and in apoptosis” (Panzer and Seegers, 1998).

The second important thesis has been also postulated while the inhibition of the tubulin polymerisation has been not observed in normal cells. Later this group extended its research and their results in another article published where the effects of Ukrain were compared with those of chelidonine and other celandine alkaloids (Panzer et al, 2000). The authors linked the effects of Ukrain and alkaloids with the regulation of mitochondrial caspases (Panzer et al, 2001). In this last article the authors reported about the inhibition of tubulin polymerisation also in transformed and normal cells and did not observed the selective effect. This paper is really a good one. However, it could be even better.

The selection of cell lines was not ideal again. No adequate normal (primary) cells were involved in experiment; from the other hand, transformed kidney monkey cell line (Vero) is not a good model in an investigation of anti-cancer drug. For example, in the 1998 study Ukrain did not exert a toxic effect on the equine lung cell line. Gene expression profiles are different in transformed and cancer cells. Haematoxylin/eosin staining technique is not so good for cell phases analysis, you can't make proper conclusion from that. But most important, authors missed one big point in their results. As clearly seen from flow cytometry experiments, especially in HELA cells after 12.5 mkg/ml 48 hrs, and less visible in WHC05 cells, after Ukrain treatment the cells quickly start to accumulate not only in mitotic phase, but larger portion undergoes apoptosis (a shift to the left side, into <1n DNA on the treated cells graphs which means that DNA is degrading). So, it is likely that affected cells (or majority of cells) rather will die. The arrested cells in other lines could die too, not necessary in the same M phase, but when they come to G0/1 phase in the next cycle, even upon Ukrain withdrawal. That what we seen in our experiments. In this sense, discussion should be interpreted in slightly different direction. Obviously, drug concentration and time treatment need to be optimized to have 100% cancer cells dead, and it could be cell line/tissue/patient dependent. But HELA cells examples they provided is really good. They just did not interpreted it right it is not about mitotic arrest, it is about apoptosis. Range of the dose used could be more gradual, especially around the point where the cells start to accumulate in G2/M phase (10-12 mkg/ml). Moreover, considering that so high doses like 50 mcg/ml are never administered in vivo, it was not really relevant to select such high Ukrain concentration for experiment. It could be concluded that presented data rather could be used as supportive evidence. The fact that drug action is reversible is quite positive from our understanding. A new experiment with the same cell lines should be carry out to clear this matter – after having concerned all comments, of course.

These studies are concentrationg on the effect on tubulin and do not regard to other effects of Ukrain and do not deny the selective effect of Ukrain on the cancer cells. The effects of ukrain on normal and malignant cells are different, this has been proofed by various research group. The last publication is by Mendoza et al, 2006.

In the tests on epidermoid carcinoma cells A431 and ME180 as well as prostate cancer cell line LNCaP, Roublevskaya et al. from Rochester University, USA revealed Ukrain to induce the G2M accumulation of cancer cells but not of normal cells. The researchers also observed an upregulation of CDK inhibitor p27 in cancer cell lines (Roublevskaya et al., 2000a, 2000b).

Cordes et al. from Eberhard-Karls-University Tubingen, Germany, investigated the effects of Ukrain on cell survival, alteration of the cell cycle and induction of apoptosis without and in combination with ionising radiation (IR). Ukrain modulated radiation toxicity of human cancer cell lines and protected normal cells from radiation. The combination of Ukrain plus IR gave enhanced toxicity in CCL-221 and U-138MG cells but not in MDA-MB-231 and PA-TU-8902 cells. A radioprotective effect was found in normal human skin and lung fibroblasts (Cordes et al, 2002).

An Italian research group, Gagliano et al. from the University of Milan, used RT-PCR, Western blot and SDS-zymography to investigate the effects of Ukrain on the expression of genes and proteins involved in the extracellular matrix remodelling associated with tumour invasion in human cultured glioblastoma cells. There was a significant, dose-related decrease in glioblastoma cell proliferation and a tendency to the downregulation of SPARC (secreted protein acidic and rich in cysteine) after treatment with Ukrain, suggesting ‘the drug may be a useful therapeutic tool for brain tumours’ (Gagliano et al, 2006).

Researchers at the Instituto Nacional de Cancerologia, Mexico City, Mexico ‘revealed that Ukrain induced apoptosis in a panel of cancer cell lines by activating the intrinsic cell death pathway. Interestingly, nontransformed fibroblasts (hTERT) cell line was insensitive to the drug.’ (Mendoza et al, 2007; used cell lines: cervical cancer HeLa, HeKB, HeKS32, HeBcl3, HeNFR and HeIKK, human colon cancer SW480, human renal carcinoma HEK293, human osteosarcoma MG-63, and immortalised human fibroblasts hTERT).

The Italian research group has extended their research in glioblastoma. In the 2007 published study authors reported that after treatment with Ukrain, glial fibrillar acidic protein (GFAP) fluorescence increased and a higher number of glioblastoma cells displayed GFAP organized into a filamentous state. Western blot analysis confirmed that Ukrain tended to upregulate this protein. Connexin 43 was not modulated by Ukrain. Ukrain induced apoptotic rate was up to 28.9%, likely mediated by cytochrome c release in the cytoplasm. (http://www.anti-cancerdrugs.com/pt/re/anticd/abstract.00001813-200707000-00006.htm; Gagliano et al, 2007).

Referenses

1. Panzer A, Seegers JC. Ukrain, a semisynthetic alkaloid of Chelidonium majus, is selectively toxic to malignant cells by causing a methapase block which results in apoptosis. Proceedings of the American Association for Cancer Research, vol. 39, March 1998.
2. Panzer A., Joubert AM, Bianchi PC, Seegers JC. The antimitotic effects of Ukrain™, a Chelidonium majus alkaloid derivative, are reversible in vitro. Cancer Letters 150 (2000), 85-92.
3. Panzer A, Hamel E, Joubert AM, Bianchi PC, Seegers JC. UkrainTM, a semisynthetic chelidonium majus alkaloid derivative, acts by inhibition of tubulin polymerization in normal and malignant cell lines. Cancer Lett., 2000 Nov 28;160(2):149-57.
4. Roublevskaia IN, Polevoda BV, Ludlow JW, Haake AR. Induced G2/M arrest and apoptosis in human epidermoid carcinoma cell lines by semisynthetic drug Ukrain. Anticancer Res. 2000 Sep-Oct;20(5A):3163-7.
5. Roublevskaia I.N., Haake A.R., Ludlow J.W., Polevoda B.V. Induced apoptosis in human prostate cancer cell line LNCaP by Ukrain. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 141-147.
6. Cordes N, Plasswilm L, Bamber M, Rodemann HP. Ukrain®, an alkaloid thiophosphoric acid derivative of Chelidonium majus L. protects human fibroblasts but not human tumour cells in vitro against ionizing radiation. Int J Radiat Biol 2002, vol. 78, No. 1, 17-27.
7. Gagliano N, Moscheni C, Torri C, Magnani I, Bertelli AAE, Nowicky W and Gioia M (2006): Effect of Ukrain on matrix metalloproteinase-2 and Secreted Protein Acidic and Rich in Cysteine (SPARC) expression in human glioblastoma cells. Anti-Cancer Drugs 17:189-194.
8. Gagliano N, Moscheni C, Torri C, Donetti E, Magnani I, Costa F, Nowicky W and Gioia M (2007): Ukrain modulates glial fibrillary acidic protein, but not connexion 43 expression, and induces apoptosis in human cultured glioblastoma cells. Anti-Cancer Drugs 2007, 18: 669-676.
9. Mendoza J., Zamora R., Gallardo J., Ceballos G., Aldana A., Espinosa M., Maldonado V., Melendez-Zajgla: NF-kappaB does not influence the induction of apoptosis by Ukrain. Cancer Biology & Therapy 5:7, July 2006: 788-793.