## 2 Pharmacological Activities
### Antitumour Properties
#### 2.6.2 Mechanism of Action of Dimeric Alkaloids
This interaction with calmodulin could explain the differences in efficacy of vinflunine that, in spite of its binds to tubulin is lower than vinblastine or vincristine, displays a better activity than vinblastine against murine tumours and human tumour xenografts. Vinflunine turned out to be a better inhibitor binding to calmodulin, therefore presents higher efficiency and lower toxicity _in vivo_[50]. A recent study based on vinorelbine supports the hypothesis about the importance of impact of \"secondary\" targets in overall action of Vinca alkaloids [51]. This could explain why, despite of very similar binding parameters on tubulin, vinorelbine and vinblastine had such a different use in clinics [52]. Keynotes Based on the Use of _C. roseus_ Plants as Biofactories for Enhancing the Production of Terpenoid Indole Alkaloids  
As stated above, the production of TIAs by _C. roseus in vitro_ cultures is a feasible technology pursued by industrial and academic interests. It is crucial to take into account, prior to the use of high producer _in vitro_ cultures of TIAs, the right choice of the _C. roseus_ cultivar which provides the best TIA levels. Although different studies based on HPLC metabolic profiling showed nearly equal amounts of TIAs in different cultivars of _C. roseus_, some _C. roseus_ cell lines produced high levels of TIAs. In fact, the selection and development of a cell line which synthesizes and accumulates TIAs depends on critical steps like the selection of appropriated explants (type, size, age, amongst others.) which are used for initiating the cell culture as well as culture conditions (nutrients, growth regulators, pH, temperature, oxygen and light, amongst others), which in turn, are critical factors on which the success of _in vitro_ cultures depends. In addition, screening of cell lines along successive subcultures [53, 54, 55] has also enabled the selection of high-yielding cell lines although they are usually unstable and tend to decrease their production after periodical subcultures. [PERSON] _et al._[56] used different cryopreservation methods, several cryoprotectants, and cooling and thawing processes. The use of mineral oil has been a useful strategy for preserving cells which restarted their growth in liquid fresh culture media [57]. On the other hand, several studies carried out using whole plants, shoot, callus and cell cultures of _C. roseus_ demonstrated that TIA production is dependent on both cell differentiation and organogenesis [58]. [PERSON] _et al._[59] observed that the pattern of TIA production in root and shoot cultures of _C. roseus_ was similar to that found in roots and leaves of whole plants. However, many TIAs cannot be synthesized by _C. roseus_ cell cultures since the activation of biosynthetic pathways is tissue-specific and developmentally regulated. [PERSON] _et al._[60] observed that levels of vindoline and catharanthine enhanced in _C. roseus in vitro_ cultures as the level of cell differentiation increased. However, these compounds were only detected in elicited conditions, the highest amounts of catharanthine and vindoline occurring in shoots (0.0039% and 0.0013% on a dry weight (DW) basis, respectively) compared with the levels found in callus (0.00019% and 0.00015% on a DW basis, respectively). The culture of hairy roots is another strategy for TIA production, since roots grow rapidly in hormone-free liquid medium, and they are composed of differentiated cells genetically stable, able to produce a broad spectrum of TIAs. In fact, the production of ajmalicine, serpentine, and catharanthine has been repeatedly reported by different authors, sometimes reaching levels superior to non-transformed root cultures (around 0.2%-0.4% DW) [61, 62]. Production of vindoline has also been detected in hairy roots, at very low levels (0.04%-0.08% DW) [61, 62, 63].