Subj: Problems with taxanes in the MTT assay

Date: 8/6/02 2:13:51 AM Pacific Daylight Time
From: European PhD Graduate Student

Dear Dr. Weisenthal!

My name is NNN NNNN. I'm a PhD-student at the pharmaceutical institute of the University of NNNN. I'm use the MTT-Assay and ATP-Assay to determine the cytotoxicity of several cytotoxic drugs. Recently I found your article "Cell Culture Drug Resistance Testing in Ovarian Cancer." In this article you wrote that it is not possible to test Docetaxel with the MTT-Assay for "different biological reasons."

Could you tell me a little bit more about this reasons or give me a hint where to find literature about this topic, because I have some problems to determine the cytotoxicity of docetaxel with MTT. I searched the internet and the library but was not able to find something about this topic.

Thank you for your help in advance.

Best regards NNN NNNN (PhD Student, Universtiy of NNNN)

From: Larry Weisenthal

We test fresh tumor specimens in DISC (membrane integrity), ATP, and MTT (mitochondrial Krebs cycle activity) assays. For most drugs, with relatively pure tumor cell populations, the results are virtually identical. In the case of paclitaxel and docetaxel, it is very common to see >80 - 90% cell death in DISC and ATP assays, but actually an INCREASE (relative to negative control minus positive control MTT formazan signal) in MTT assay.

For example DISC 12% of control cell survival (88% cell death)
ATP 16% of control ATP content (84% cell death)
MTT 145% of control formazan signal (negative control minus positive control)

What is probably happening is that there is actually an increase in the number or metabolic activity of mitochondria of the surviving cells, even in cases where the majority of the cells are being killed by taxanes. Similar observations have been made by others, e.g.

1: Eur J Cancer 1993;29A(11):1573-7 Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival. Pagliacci MC, Spinozzi F, Migliorati G, Fumi G, Smacchia M, Grignani F, Riccardi C, Nicoletti I. Istituto di Clinica Medica 1, Perugia University School of Medicine, Italy.

The natural isoflavone genistein inhibits the growth of a number of tumour cell lines in vitro. During investigations on the antiproliferative effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) colorimetric assay consistently underestimated the growth inhibitory activity of the substance. Cell proliferation was markedly inhibited by genistein in three tumour cell lines (MCF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929, mouse transformed fibroblasts) when cell number was evaluated by direct counting, whereas a 72-h MTT assay failed to reveal any growth-inhibitory effect. Cell cycle analysis by propidium iodide staining and flow-cytometry revealed a G2/M cell cycle arrest after genistein treatment. Genistein-treated cells displayed an increase in cell volume and in mitochondrial number and/or activity, as revealed by enhanced formazan generation and increased uptake of the vital mitochondrial dye rhodamine 123. These results suggest that alterations in cell cycle phase redistribution of tumour cells by genistein may significantly influence mitochondrial number and/or function and, consequently, MTT reduction to formazan. This may constitute an important bias in analysing the effects of genistein, and possibly other drugs that block the G2/M transition, on growth and viability of cancer cells in vitro by MTT assay.

2: Eur J Cancer 1993;29A(11):1502-3 Comment on: Eur J Cancer. 1993;29A(11):1573-7. In vitro assays for antitumour activity: more pitfalls to come? Hanauske AR. I. Department of Medicine, Klinikum rechts der Isar der Technischen Universitat Munchen, F.R.G.

Additional comments from Larry Weisenthal:

This latter paper was an editorial about the first paper. As I recall (and I don't have Hanauske's editorial here in front of me), the editorial made mention of Hanauske's personal observation that the same phenomenon applied also to taxanes.

If you want to study taxanes, my recommendation is NOT to use the MTT assay, but instead to use an assay like the ATP assay, DISC assay, and/or the fluoroscein diacetate assay. e.g.

1: Cancer 1997 Mar 15;79(6):1225-33 Differential activity of Cremophor EL and paclitaxel in patients' tumor cells and human carcinoma cell lines in vitro. Csoka K, Dhar S, Fridborg H, Larsson R, Nygren P. Division of Clinical Pharmacology, Uppsala University, Sweden.

BACKGROUND: Previous studies indicate that Cremophor EL (CEL), the excipient for Taxol, a clinical preparation of paclitaxel, has biologic properties per se. METHODS: The cytotoxic activity of Taxol and its solvents CEL/ethanol, paclitaxel in ethanol, and 14 other cytotoxic drugs was investigated in vitro in 10 human carcinoma cell lines and 183 tumor samples from patients with tumors of various types. Cytotoxicity was determined by the fluorometric microculture cytotoxicity assay. RESULTS: In the cell lines, Taxol was generally more active than paclitaxel; this may have been due to an additive effect of the diluent. This activity was pronounced in sublines expressing tubulin-associated and P-glycoprotein-mediated drug resistance, indicating involvement of these mechanisms in paclitaxel resistance and their modulation by CEL. Taxol and paclitaxel were highly cross-resistant to other tubulin-active agents, whereas the low cytotoxic effect of CEL seemed unrelated to other drugs. In the samples from patients, Taxol was less active than in the cell lines but showed a differential activity that corresponded reasonably well with that in the clinic. CEL and Taxol were similarly active, indicating that paclitaxel did not add substantially to the activity of Taxol. CONCLUSIONS: Whereas the cell line data clearly confirmed the well-known properties of paclitaxel, a more valid model using tumor cells from patients demonstrated that CEL significantly contributes to the efficacy of Taxol in vitro. The clinical relevance of this finding remains to be elucidated.

>>> Best wishes, Larry Weisenthal Huntington Beach, CA, USA