HPLC and HR-LC/MS analysis confirmed the presence of Exo-MS with some impurities. NMR technique not being sensitive, low concentrations of Exo-MS could not allow a clear correlation in TOCSY, though threonine, β-alanine and γ1β-ornithine could be deciphered.
HPLC analysis of ferri-MBS indicated about 98% purity. When the siderophore is complexed with iron, the red colored complex shows maximum absorbance at 450 nm, whereas the peptide bonds of MBS have an absorbance maximum at 220 nm. The peaks seen at A
220nm corresponded with the peaks observed at A
450 nm indicating pure MBS preparation. These were similar to the peaks obtained for MBS by Ratledge and Ewing [
13].
Only one peak was obtained in the HPLC analysis of ferrioxamine-B indicating it was pure.
To be used for iron-depletion from microbial environments, siderophores need to be in a desferri form and not saturated with iron. Therefore, desferri form of siderophores were obtained using the isolation protocols for Exo-MS and MBS, but without the addition of iron during the entire isolation process. Iron was added to small aliquots only for determining purity and concentration of the siderophores.
Effect of Exo-MS, MBS, and DFO-B on mammalian cell lines
Non-malignant cells and malignant cells lines were used to study the effect of the iron-chelators on mammalian cells. The exposure with the siderophores was done for 18–24 h because the average time of division for the mammalian cells in vitro is 18–28 h. Besides, siderophores are not respiratory inhibitors, and are expected to affect proliferation of cells. When some pathogenic bacteria were treated with these siderophores for 18–24 h, they were inhibited (manuscript under preparation). This was also taken into consideration whilst subjecting mammalian cells to siderophores for 18–24 h.
The experiments with mammalian cells showed that concentrations of Exo-MS and DFO-B that significantly inhibited the murine cancer cell line RAW 264.7 were not inhibitory to the normal cell line NIH/3 T3. It should be noted that both Exo-MS and DFO-B had no significant effect on the non-malignant human cell line HEK 293 up to 0.5 mg/mL and 1 mg/mL, respectively.
DFO-B also inhibited the proliferation of human breast cancer (MCF-7) and human leukemia (K 562) cell lines. However, it had no effect on human liver cancer cell line (HEPG2) even at 10 mg/mL. Exo-MS did not inhibit any of the human cancer cell lines tested. DFO-B has already been shown to have anti-proliferative effect on cancer cells [
14], and hence was used here as a control.
When ferri-siderophores were evaluated for activity using the same MTT assay, no inhibitory effect on the cell proliferation was observed (data not shown). In this form, the siderophores were already saturated with iron, and therefore, could not deprive the cells of iron.
Earlier, some investigators have reported that a lipid-soluble siderophore from
Mycobacterium tuberculosis (Mtb), mislabelled by the authors as (desferri-)exochelin 772SM, induces death by apoptosis in human breast cancer cells without harming normal breast epithelial cells [
15]. However, the correct term for the siderophore referred to by the authors should have been ‘Carboxymycobactin’, since water-soluble exochelin is not produced by
Mtb.
Hoke et al. reported that DFO-B could be used as an adjunct to chemotherapy along with doxorubicin drug to inhibit breast tumor growth without any cardiotoxicity [
16]. DFO-B was also found to be cytotoxic to malignant cells of neural origin [
17,
18]. When DFO-B was given during the initial stages of tumor formation, it resulted in either regression or slower tumor growth due to decrease in intracellular Fe
3+ concentration [
19]. DFO-B was found to be inhibitory to leukemia cells in vivo as evident from the reduction in the cell numbers [
20].
Since the water-soluble siderophores Exo-MS and DFO-B had no effect on HEPG2 even at high concentrations, effect of the lipid-soluble siderophore MBS was evaluated on HEPG2. MBS significantly decreased the percent survival of HEPG2 cells at very low concentrations (10–20 μg/mL). This concentration is almost 50–1000 times lower than Exo-MS and DFO-B concentrations that inhibited RAW 264.7 cell line. Due to its lipophilic nature, MBS may traverse eukaryotic cell membrane. This lipid-soluble characteristic makes MBS much more efficient at lower concentrations in trapping intracellular iron than the hydrophilic siderophores. Since very low concentrations of MBS showed anti-proliferative activity in vitro, it would be interesting to investigate its effects in vivo.
In the light of our results with the lipid-soluble MBS, conjugating water-soluble siderophores with lipid molecules may make them more effective against tumor cells. These siderophore conjugates could be evaluated as anticancer agents by themselves, or along with other therapies.