The data presented here show that normal epithelial cells can tolerate higher concentration of PTX without apparent harm than HNSCC cells. The effect of PTX shown on tumor cells suggests that their morphology can be used as an index of PTX toxicity. Morphological change in tumor cells also correlated with LDH release indicating a loss of cellular function, primarily the membrane integrity as would be expected in response to PTX which is known to affect the plasma membrane. It is obvious that many of the pharmacological effects of PTX are attributable to the effect of this substance on trans-membrane ion transfer. PTX has a unique action on the Na+,K+-ATPase, converting the pump into an ion channel and resulting in K(+) efflux, Na(+) influx and membrane depolarization. PTX can in vitro cause lysis of mouse spleen cells which has been attributed to a PTX-induced increase in cellular calcium levels.
The toxicity of PTX in mammals is strongly dependent upon the route of administration. PTX is most toxic by intra venous (iv) injection, the LD50 in mice amounted to 0.15 -0.53 μg/kg[1, 30]. The PTX toxicity by ip administration is lower than that by iv injection, with values of 0.31-1.5 μg/kg being reported for mice[31, 32]. PTX is much less toxic orally than after iv or ip administration. Results from the few existing studies reports an oral LD50 from 510 μg/kg to 767 μg/kg in mice[33, 34].
PTX has been described as a tumor promoter[20, 21]. This might misleadingly suggest that it is capable of causing tumors. Therefore it is important to note that the basis to classify an agent as a tumor promoter is conditional and is performed only within the context of a two-stage (or multistage) model protocol. The tumor-promoting activity of PTX has been investigated earlier using mouse skin. Thereby, in the first stage carcinogenesis was initiated with the mutagenic compound 7,12-dimethylbenz[a]anthracene (DMBA). In the second stage, repeated application of PTX was performed over a period of several weeks. In mice treated with DMBA and PTX tumor development occurred, but no tumors were observed in animals treated with PTX alone suggesting that PTX treatment alone is not sufficient to generate tumors. To verify this, we performed long-time experiments in which a group of mice were treated daily with 0.5ng PTX for 5 days. By using this low PTX concentration we based our approach on results which showed that PTX concentrations higher than 0.5ng are already toxic to mice. The animals were observed over a period of 8 months without finding evidence of tumor development. Also other studies showed that PTX does not act as a tumor initiator in a Balb/c 3T3 cell transformation assay and it was negative in the Ames mutagenecity test using different strains (TA98, TA100, TA102 and TA1537). Based on these findings we used PTX to treat tumor xenografts established in SCID mice. Treating these mice with doses as little as 68-83ng/ kg bodyweight we observed rapid and progressive tumor destruction without recognizing any apparent disease symptoms. However, this was only the case when PTX was admistisred intratumoral. None of the mice did show any undesired pattern of behavior during therapy nor during a follow up period of 2 weeks, suggesting that low doses of intratumoral injected PTX might even be beneficial, due to them selectively killing tumor cells rather than normal epithelial cells, but no effects were seen after ip PTX injections.
Alterations in ion gradients induced by PTX at the plasma membrane level play a crucial role in cytotoxic and cell death events. Experimental studies indicated that PTX targets the Na+, K+ ATPase, and thereby destroys the ion gradient [5,9,12,]. This may lead to a lack of Na+, K+ ATPase causing dramatic effects on cell function. It is reasonable to hypothesize that a response of the cells to this external influence is the post-production of Na+, K+ ATPase in order to replace the quantity indispensible for stable cellular conditions. To demonstrate this we analyzed the transcriptional activity of several genes and found that treatment of cells with PTX in fact influences the expression of the ATP1AL1 gene that encodes the Na+, K+ ATPase. The initial down-regulation and the subsequent progressive up-regulation of this gene is a typical phenomenon of self-regulating, self-protection processes i.e. the ability of the cells to maintain their internal equilibrium due to PTX as an external influencing factor. PTX on the other hand seems to influence the energy metabolism of the cells since we have shown that GAPDH gene expression was also down –and up-regulated as a function of PTX concentration. The expression profiles for both ATP1AL1 and GAPDH genes suggest that PTX induces in the cell lines studied both transcriptional gene suppression and activation. The mechanism involved in such bidirectional transcription process is poorly defined. Recent observations suggest that bidirectional transcription in human cells is an endogenous gene regulatory mechanism whereby small non-coding RNA mediated transcriptional regulation can act in both suppressive and activating manner.
PTX stimulates JNK activation through a pathway that involves ion flux. Initial studies showed that PTX affects JNK activation through a mechanism that involves sodium influx. A later study conducted in rat fibroblasts suggested that PTX stimulates JNK activation through a mechanism that involves potassium efflux. It was also demonstrated that PTX-stimulated signals are transmitted to JNK through the activation of a protein kinase cascade, so that the induction of ion flux by PTX results in the activation of MEK4 (MAPK kinase 4) which phosphorylates and activates JNK[42–45]. Collectively, the JNK MAPKs as an evolutionarily-conserved family appear to be important mediators of PTX-stimulated signals. Noteworthy in this regard is the involvement of JNK3 (MAPK10) in these signaling events and has been verified by our JNK3 protein kinase inhibition experiment showing that the repression of the JNK3 expression is essential for the enhancement of PTX toxicity in cancer cells.
In conclusion, we have demonstrated that head and neck cancer cells and xenografts are more sensitive to PTX than normal cells. Because PTX binds to cell surface receptors present on malignant and benign cells, and acts more effectively upon HNSCC cells, there is a need to pay more attention to this natural product to further define the way of its optimal potential use which may extend our knowledge of the biology of head and neck cancer.