Salinomycin is a molecule produced by terrestrial bacteria of the species Streptomyces albus. It was previously known that this molecule acts selectively against cancer stem cells. Salinomycin has been shown to kill breast cancer stem cells in mice at least 100 times more effectively than the anti-cancer drug paclitaxel. The mechanism of action by which salinomycin kills cancer stem cells remains unknown, but is thought to be due to its action as a potassium ionophore due to the detection of nigericin in the same compound screen.
Now, researchers from Lund University have created a fluorescent variant of salinomycin to understand how it works. Capturing images of when the molecule enters a cell has enabled the researchers, using cell-biological methods, to successfully describe how and where the molecule counteracts the cancer stem cells. An active fluorescent salinomycin conjugate reveals rapid cellular uptake and strong localization to the endoplasmic reticulum. Ionophore activity in this organelle is connected to phenotype effects.
Synthesis and Cellular Uptake of Fluorescent Salinomycin Conjugates
To visualize cellular uptake and subcellular localization of salinomycin, a fluorescent conjugate that was functionally equivalent to the native structure is needed to design. Selective ligation of fluorophores to complex natural products like salinomycin without impairing their properties is nontrivial.
Experiments demonstrated that ligation of a nitrobenzoxadiazole(NBD) reporter to the C20 hydroxyl of salinomycin gives a fluorescent conjugate that is functionally equivalent to salinomycin and hence suitable for mechanistic investigations. The real-time uptake experiments moreover revealed salinomycin derivatives entering cells on a time scale that supports using such structures for acute cell experiments.
Salinomycin Accumulates in the Endoplasmic Reticulum and in Lipid Droplets
The subcellular localization of salinomycin conjugates in breast cancer cells was investigated using confocal microscopy.
LDs are lipophilic structures that originate from lipid deposits in the ER phospholipid bilayer and thus share many of its characteristics. They interpret the preferential accumulation of the conjugates in the ER and LDs as a reflection of their lipophilic nature. Combined with the observation that only salinomycin derivatives capable of electroneutral alkali metal ion transport induces phenotype effects, the localization data indicate that the principal function of salinomycin is ionophore activity in the ER membrane.
Salinomycin Induces ER Ca2+ Release, ER Stress, and PKC Activation
Accumulation of salinomycin in the ER suggests that its ion transport properties may underlie its effect on the cytosolic Ca2+ concentration. The Ca2+ source contributing to the increase in cytosolic Ca2+ caused by salinomycin was thus investigated.
Combined, the data show that salinomycin causes an increase in the release of Ca2+ from the ER. This release ultimately results in an increase in CHOP expression and activation of calcium-dependent PKC, both known factors contributing to inhibition of Wnt signaling.
Scientists developed a fluorescent NBD conjugate of salinomycin that retains the activity profile of the native structure. This conjugate was used to guide a mechanistic investigation of the molecular basis for the activity of salinomycin against stemlike cancer cells. The conjugate was shown to rapidly enter breast cancer cells and localize in the ER and LDs. Uptake of salinomycin into the ER was then shown to result in an enhanced Ca2+ release from this organelle, presumably a result of a counter transport of K+ by salinomycin. Depletion of Ca2+ from the ER led to ER stress and activation of the UPR, which induced up-regulation of CHOP. The concomitant increase in cytosolic Ca2+ caused activation of conventional PKCs. Since both up-regulation of CHOP and activation of PKC inhibit the Wnt signaling pathway, they connects the mechanism of salinomycin at the molecular level to previously described phenotype effects.
The research results may contribute new approaches to the development of cancer drugs both for treatment of cancer and for reducing the risk of relapse.
More information, please click https://www.lunduniversity.lu.se/article/fluorescent-molecules-reveal-how-cancer-stem-cells-are-selectively-inhibited