Starting with the scientific program we first dealt with the "master question" of the cancer situation, namely,
1. Why are tumors and metastasis not attacked by the immune system?
2. Why are they able to coexist with the normal tissue?
The observation that tumors being generally acidic leads to the idea that the acidity could be involved or could be the key for answering these questions. Could it be that immune reactions are pH-dependent?
We started a comprehensive study to obtain information on this topic. And in fact, it turned out that indeed the known kill reactions of the immune system are all pH dependent, which means that the kill reactions cease to occur with growing acidity (see literature). There are also indications that lactic acid plays a role. So, it is likely to assume that the acidity of tumor is an archaic, extremely effective shield for protection against immune attack. And indeed, it is an amazing effect that it makes no difference how strong the immune attack is, it will never succeed. In this respect it is worth mentioning that artificial stimulation, e.g. with vaccines, will never succeed.
The acidic milieu of cancer cells may have a further advantage for surviving. Lytic enzymes, which are active in acidic milieu, enable nutrition by penetrating neighbor tissue, thereby becoming extensively independent of blood supply. Reducing blood supply by angiogenesis inhibitors, should not bring the aimed success, on the contrary, it may induce stimulation for metastasis formation, in order to survive in host tissues.
The research work started with a comprehensive screening procedure in order to find the appropriate compound. To select pH-sensitive compounds, every compound envisaged was tested by 5 procedures, namely by:
- Proliferation assay (3H-Thymidin)
- Pore formation assay (Propidium Iodide FACS-Analysis)
- Inhibition of respiration chain (XTT-assay)
- Lactate transport assay
- Measurement of Apoptosis
The evaluation was proceeded with 8 human tumor cell lines (RT112, Capan1, SK-MEL-30, MCF-7, HepG2, LS1 74T, Colo-699, SK-N-IVIC). In addition, potential compounds were tested in xenograph models (human tumors growing subcutaneously (s.c.) on nude mice).
The very surprising outcome was that only 5 salicylates (derivatives of salicylic acids) showed potent pH-dependent effects as desired. And what was further remarkable, 3 of them were approved drugs, namely Diflunisal, PAS and Aspirin.
Four pH-dependent actions on cancer cells may be demonstrated by the following in vitro experiments of figures 1-4.
In Fig. 1 the pH-dependent action of the mitochondrial respiration chain is presented in normal state (control) and by action of salicylates. From the depicted graphs it is evident that by lowering the pH below 7, by action of salicylates the respiratory chain ceases to function. Within the physiological range of pH > 7.0 the respiration is not affected by salicylates. This toxicity effect is predominantly caused by Diflunisal.
In Fig. 2 an example of pore-formation is presented. Pore-formation is proceeded in two different pH characteristics, one running at pH < 6.5 and another at pH < 7.0, depending on the salicylates concentrations. The actions of ASA and PAS are only observed at pH < 6.5. It is only Diflunisal at special concentrations that is able to induce poration at pH < 7.0.
The experiments of the figures 1 and 2 were performed in absence of albumin. If albumin is added, a situation which corresponds to the physiological conditions, the efficacy of the salicylates changes drastically. The activity of Diflunisal is canceled whereas that of ASA and PAS is less drastically changed.
What is of decisive importance is the observation demonstrated in figures 3 and 4 that the action of Diflunisal in the presence of albumin can be restored if Diflunisal is applied together with ASA or PAS. An effect that we call "synergistic action of ASA and PAS on Diflunisal". Synergistic acting combinations of Diflunisal-ASA or Diflunisal-PAS, therefore, enable Diflunisal to act under physiological conditions.
In Fig.3 this behavior is demonstrated in case of the respiratory chain compared to Fig.1. In the presence of albumin Diflu and ASA only show efficiency at pH < 6.7. If the 2 compounds are combined the action becomes restored comparable to Diflu solo in Fig.1.
The same behavior can be observed testing the proliferation of cancer cells in Fig.4. The compounds solely applied behave like the control proliferation. In combination, however, proliferation is cancelled up to pH < 6.9.
The canceling of Diflu action by albumin, obviously, is due to an almost 100% binding of Diflunisal onto albumin and the synergistic action of ASA or PAS may be understood as an activation of Diflu despite of binding onto albumin. However, the Diflunisal thereby is not set free as could be thought at first sight, but the activating mechanisms are indeed much more complex.
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© 2009 Prof. Dr. W. Kreutz, Krozingerstr. 3 (Am Schlossberg), 79214 Staufen, Germany