## 6 Biological Activity of the Hapalindole-Type Alkaloids
### Biochemical, Molecular and Cellular Bioactivity
Interestingly, studies of the antimitotic activity of ambiguine D in a plant cell model, likewise, demonstrated elevated ROS, and associated increases in lipid peroxidation, and was suggested to play a role in the observed cell death [66]. Moreover, immunoblotting studies of breast cancer cells treated with ambiguine I showed down-regulation of both p65 and p50 subunits of NF-\(\kappa\)B, and the upstream kinase, IKK\(\kappa\); in turn, ambiguine I treatment led to a down-regulation of intracellular cell adhesion protein, ICAM-1, which is involved in migration and invasion of cancer cells, suggesting a potential novel mechanism of the compound as an anticancer drug [65]. As discussed above, a considerable amount of attention has been placed on the welwitindolinones with respect to antiproliferative--and potential anticancer--activity. Although much of this research has focused on synthetic studies, the original characterization of the molecules and their biological activity identified compelling mechanistic details [67; 68]. Specifically, welwitindolinones were isolated as part of studies focused on the identification of compounds which reverse MDR of cancer cells. Two variants, welwitindolinone C isothicyanate and its \(N\)-methyl derivative, were specifically shown to abate acquired drug-resistance of a breast cancer cell line (_i.e._, MCF-7/ADR) with respect to a diverse range of anticancer drugs, including taxol, vinblastine, colchicine, daunomycin and actinomycin D, at sub-micromolar concentrations [68]. In particular, \(N\)-methylwellwitindolinone C isothicyanate showed pronounced activity (at concentrations below that of the described MDR-reversing drug, verapamil). And notably, the isonitrile variant of welwitindolinone C showed no activity, suggesting role of the isothicyanate functional group. Similarly, accumulation of radiolabeled [3H]-taxol was observed in an MDR variant of an ovarian carcinoma cell line (_i.e._, SK-VBL-1), and paralleled the potency of MDR-reversing potential of the variants in breast cancer cells; interestingly, however, the \(N\)-methyl derivative was not found to lead to the accumulation [\({}^{3}\)H]-taxol in treated cells, whereas the non-methylated wellwitindolinone C isothicyante and verapamil did. These studies, moreover, elucidate an apparent interaction with the P-glycoprotein (_i.e._, ATP-binding efflux pump) pathway of MDR. Furthering the interest in these two compounds, a subsequent study demonstrated, in addition to MDR-reversing activity, antiproliferative activity of the wellwistatins themselves, and specifically, interference with microtubule formation via inhibition of tubulin polymerization [67]. With respect to the haplindoles, studies by [PERSON] _et al._[62; 64] have pointed to inhibition of RNA polymerase as a possible mode of action. Investigating allebotathic potential, it was shown that 12-_epi_-hapalindole E inhibited the growth of a wide range of bacteria, fungi and algae, and more specifically, in a Gram-positive bacterial model (_i.e._, _Bacillus subtilis_ 168), inhibited both RNA and protein synthesis [62]. It was further demonstrated _in vitro_ that the haplindole inhibited RNA polymerase directly (rather than interacting with the DNA template). And subsequent studies using Gram-negative _E. coli_ RNA polymerase both confirmed the direct interaction with the enzyme (and not DNA), and also demonstrated several notable difference from binding activity of other RNA polymerase inhibiting antibiotics, _i.e._, rifampicin [64]. The study concluded, however, based on the relatively high concentration required for RNA polymerase inhibition, that additional mechanisms were likely involved in the antimicrobial activity of the haplindoles. Interestingly, several haplindoles--including the tricyclic haplindoles E and C isonitrile, and tetracyclic haplindole L--were recently shown to modulate sodium channels in a neuroblastoma cell line [59].