Dodecyl-TPP Targets Mitochondria and Potently Eradicates Cancer Stem Cells (CSCs): Synergy With FDA-Approved Drugs and Natural Compounds (Vitamin C and Berberine)

Abstract

Elevated mitochondrial biogenesis and/or metabolism are distinguishing features of cancer cells, as well as Cancer Stem Cells (CSCs), which are involved in tumor initiation, metastatic dissemination, and therapy resistance. In fact, mitochondria-impairing agents can be used to hamper CSCs maintenance and propagation, toward better control of neoplastic disease. Tri-Phenyl-Phosphonium (TPP)-based mitochondrially-targeted compounds are small non-toxic and biologically active molecules that are delivered to and accumulated within the mitochondria of living cells. Therefore, TPP-derivatives may represent potentially “powerful” candidates to block CSCs. Here, we evaluate the metabolic and biological effects induced by the TPP-derivative, termed Dodecyl-TPP (d-TPP) on breast cancer cells. By employing the 3D mammosphere assay in MCF-7 cells, we demonstrate that treatment with d-TPP dose-dependently inhibits the propagation of breast CSCs in suspension. Also, d-TPP targets adherent “bulk” cancer cells, by decreasing MCF-7 cell viability. The analysis of metabolic flux using Seahorse Xfe96 revealed that d-TPP potently inhibits the mitochondrial oxygen consumption rate (OCR), while simultaneously shifting cell metabolism toward glycolysis. Thereafter, we exploited this ATP depletion phenotype and strict metabolic dependency on glycolysis to eradicate the residual glycolytic CSC population, by using additional metabolic stressors. More specifically, we applied a combination strategy based on treatment with d-TPP, in the presence of a selected panel of natural and synthetic compounds, some of which are FDA-approved, that are known to behave as glycolysis (Vitamin C, 2-Deoxy-Glucose) and OXPHOS (Doxycyline, Niclosamide, Berberine) inhibitors. This two-hit scheme effectively decreased CSC propagation, at concentrations of d-TPP toxic only for cancer cells, but not for normal cells, as evidenced using normal human fibroblasts (hTERT-BJ1) as a reference point. Taken together, d-TPP halts CSCs propagation and targets “bulk” cancer cells, without eliciting the relevant undesirable off-target effects in normal cells. These observations pave the way for further exploring the potential of TPP-based derivatives in cancer therapy. Moreover, TPP-based compounds should be investigated for their potential to discriminate between “normal” and “malignant” mitochondria, suggesting that distinct biochemical, and metabolic changes in these organelles could precede specific normal or pathological phenotypes. Lastly, our data validate the manipulation of the energetic machinery as useful tool to eradicate CSCs.

Combination Therapy with Vitamin C Could Eradicate Cancer Stem Cells

“…In addition, Ferrous (Fe3+) ion reduces to ferric (Fe 2+ ) ion and enters the CSCs as Fe 2+ and hydrogen peroxide (H2O2), thereby increasing the ROS production. Studies have shown the doxycyclin [117,120], azithromycin [20] and dodecyl tri-phenyl-phosphonium (dTPP) [121] inhibit mitochondrial activity which in turn leads to Vit.C induced cell death.…”

“…A recently published article reemphasized the effect of Vit.C on eradicating CSCs. The effect of the integrated metabolic strategy to exterminate CSCs was validated [121]. Breast cancer cells MCF-7 and MDA-MB-231 were treated with mitochondria-a targeted small non-toxic compound Tri-Phenyl-Phosphonium (TPP) derivative called Dodecyl-TPP (d-TPP) ( Figure 2).…”

“…However, it was also observed that not only the d-TPP switches the energy pathway in these breast cancer cell lines, but also the sensitivity to other metabolic inhibitors like Vit.C and 2-DG (glycolysis inhibitors) and Doxycycline, Niclosamide, and Berberine (OXPHOS inhibitors). Thus this study conferred a synergistic effect, a "Two-Hit" approach by metabolic inhibitors on the CSC propagation, and has proven that Vit.C is a potential compound to target the glycolysis pathway for this effect on CSCs [121].…”

The Metabolic Heterogeneity and Flexibility of Cancer Stem Cells

“…At the least, experiments using model mice have suggested that overeating after calorie restriction promotes the proliferation of CSCs and distant metastases [ 190 ]. On the other hand, some food-derived ingredients, e.g., eicosapentaenoic acids and docosahexaenoic acids, which are omega-3 polyunsaturated fatty acids, and vitamin C, have a therapeutic effect on CSCs [ 191 , 192 , 193 , 194 ]. However, there have been no reported clinical cases in which these components were effective for tumor regression.…”

Complex Mitochondrial Dysfunction Induced by TPP+-Gentisic Acid and Mitochondrial Translation Inhibition by Doxycycline Evokes Synergistic Lethality in Breast Cancer Cells

“…At 15 min of exposure, GA-TPP + C 10 inhibited only Complex I-dependent respiration ( Figure 3C). Given that alkyltriphenylphosphonium cations decrease mitochondrial OCR through ETC inhibition [37][38][39] and that p-hydroquinones, similar to GA, inhibit Complex I-dependent respiration [10,28], we compared the inhibitory efficacy on Complex I-dependent respiration by using GA-TPP + C 10 with GA and (10-hydroxydecyl) triphenylphosphonium bromide (OH-C 10 TPP + ), both chemical fragments that constitute the mitochondria-targeted studied compound. As shown in Figure 3E, OH-C 10 TPP + and GA had much lower inhibitory efficacy against rotenone-sensitive respiration than GA-TPP + C 10 , suggesting that this compound exhibits improved activity on the mitochondrial bioenergetics of cancer cells.…”