Doxycycline, Azithromycin and Vitamin C (DAV): A potent combination therapy for targeting mitochondria and eradicating cancer stem cells (CSCs)

Here, we devised a new strategy for eradicating cancer stem cells (CSCs), via a “synthetic-metabolic approach, involving two FDA-approved antibiotics and a dietary vitamin supplement. This approach was designed to induce a “rho-zero-like phenotype in cancer cells. This strategy effectively results in the synergistic eradication of CSCs, using vanishingly small quantities of two antibiotics. The 2 metabolic targets are i) the large mitochondrial ribosome and ii) the small mitochondrial ribosome. Azithromycin inhibits the large mitochondrial ribosome as an off-target side-effect. In addition, Doxycycline inhibits the small mitochondrial ribosome as an off-target side-effect. Vitamin C acts as a mild pro-oxidant, which can produce free radicals and, as a consequence, induces mitochondrial biogenesis. Remarkably, treatment with a combination of Doxycycline (1 μM), Azithromycin (1 μM) plus Vitamin C (250 μM) very potently inhibited CSC propagation by >90%, using the MCF7 ER(+) breast cancer cell line as a model system. The strong inhibitory effects of this DAV triple combination therapy on mitochondrial oxygen consumption and ATP production were directly validated using metabolic flux analysis. Therefore, the induction of mitochondrial biogenesis due to mild oxidative stress, coupled with inhibition of mitochondrial protein translation, may be a new promising therapeutic anti-cancer strategy. Consistent with these assertions, Vitamin C is known to be highly concentrated within mitochondria, by a specific transporter, namely SVCT2, in a sodium-coupled manner. Also, the concentrations of antibiotics used here represent sub-antimicrobial levels of Doxycycline and Azithromycin, thereby avoiding the potential problems associated with antibiotic resistance. Finally, we also discuss possible implications for improving health-span and life-span, as Azithromycin is an anti-aging drug that behaves as a senolytic, which selectively kills and removes senescent fibroblasts.

Combining two complementary inhibitors of mitochondrial protein translation at a low-dose: Doxycycline and Azithromycin

Here, we experimentally evaluated if the inhibitory effect of Doxycycline on mammosphere formation could be potentiated, by a combination with Azithromycin. To this end, Doxycycline and Azithromycin were tested alone or in combination at low concentrations.

shows that at low concentrations (0.1 μM and 1 μM) Doxycycline and Azithromycin alone had little or no effect on the inhibition of mammosphere formation. However, the combination of 1 μM Doxycycline and 1 μM Azithromycin exerted a very significant inhibitory effect on mammosphere formation.

Note that in combination, Doxycycline and Azithromycin display a marked increased efficacy in the inhibition of mammosphere formation, relative to when the drugs are used alone (the IC-50 for the combination is lower than for Azithromycin alone and lower than for Doxycycline alone, when both agents are used individually). These results suggest that a combination of Doxycycline and Azithromycin might have more therapeutic efficacy, than the two drugs used alone.

To evaluate if the inhibitory effects on mammosphere formation of this double combination is related to mitochondrial function, we next examined the metabolic profile of MCF7 cell monolayers pre-treated with the combination of 1 μM Doxycycline and 1 μM Azithromycin or with the same drugs alone for 3-days. Interestingly, the rates of both oxidative mitochondrial metabolism and glycolysis were significantly reduced by the DOX-AZI combination pre-treatment, as assessed using the Seahorse XFe96 analyzer. This resulted in significant reductions in respiration (basal and maximal), as well as reduced ATP levels (). Similarly, both glycolysis and glycolytic reserve were decreased by the DOX-AZI combination (). Finally, as seen in , MCF7 cancer cells were shifted from a highly energetic profile to a metabolically quiescent state. However, the combination of 1 μM Doxycycline with 1 μM Azithromycin is initially non-toxic under anchorage-independent growth conditions ().

Taken together, these results indicate that the combination of low-dose Doxycycline with Azithromycin might be a more efficacious therapeutic option than Doxycycline alone for the eradication of CSCs, by inhibition of mitochondrial function and glycolysis.

Combining two inhibitors of mitochondrial protein translation, with a pro-oxidant: Doxycycline and Azithromycin Plus Vitamin C (DAV)

Our results indicate that a combination of Doxycycline and Azithromycin is more efficacious than the individual antibiotics in inhibiting CSCs propagation. Thus, we sought to test the hypothesis that a triple combination of Doxycycline, Azithromycin and Vitamin C could more potently inhibit CSC propagation. We have previously shown that Vitamin C alone inhibits CSC propagation with an IC-50 of ~1 mM [6].

Importantly, simultaneous treatment with 1 μM Doxycycline, 1 μM Azithromycin and 250 μM Vitamin C very potently inhibited CSC propagation by ~90% (). Thus, near complete ablation of 3D tumor-sphere forming ability was achieved at very low concentrations of these two antibiotics, suggesting that this DAV triple combination targets CSCs at a vulnerable weak point.

Next, we evaluated the potential inhibitory effects of the DAV triple combination on mitochondrial function, by determining the metabolic profile of MCF7 cell monolayers pre-treated with a combination of 1 μM Doxycycline, 1 μM Azithromycin and 250 μM Vitamin C for 3-days. Remarkably, the rate of oxidative mitochondrial metabolism was reduced by >50% and ATP levels were drastically reduced (by >90%), as assessed using the Seahorse XFe96 analyzer. Overall, this resulted in significant reductions in both basal and maximal respiration (). In contrast, glycolysis was increased, while glycolytic reserve was decreased by the DAV triple combination ().

Finally, as seen in , treatment with 250 μM Vitamin C alone, significantly increased both mitochondrial metabolism and glycolysis in MCF7 cancer cells. These observations are consistent with the idea that Vitamin C alone acts as a mild pro-oxidant and stimulates mitochondrial biogenesis, driving increased mitochondrial metabolism and elevated ATP production. This interpretation is consistent with our experimental data directly showing that the inclusion of two inhibitors of mitochondrial protein translation with Vitamin C, blocks and completely reverses this Vitamin C induced increase in mitochondrial oxidative metabolism ().

Taken together, our evidence supports a novel combined metabolic strategy to better eradicate CSCs. More specifically, we demonstrate that the inhibitory effects of Doxycycline on the CSC population can be potentiated by combination with another FDA-approved antibiotic, Azithromycin, and a dietary supplement, namely Vitamin C (a mild pro-oxidant).

This new DAV therapeutic strategy should provide for the more efficient eradication of CSCs. We aim to test this hypothesis in future clinical trials.

Evaluating the temporal Effects of pre-treatments on the efficacy of the DAV triple combination, using CSC propagation as a read-out

We devised a system to test whether it was required for all three components of the DAV triple combination to be administered at the same time, by using a pre-treatment strategy, prior to initiating the 3D mammosphere stem cell assay.

Briefly, MCF7 cells, grown as monolayer cultures, were first pre-treated with either Vitamin C alone (250 μM), or Doxycycline Plus Azithromycin (D + A; 1 μM each), for a period of 7 days. Then, MCF7 cells were harvested with trypsin and re-plated under anchorage-independent growth conditions, in the presence of various combinations of Vitamin C, Doxycycline and Azithromycin.

shows that 7 days of pre-treatment with either Vitamin C alone or Doxycycline Plus Azithromycin (D + A), rendered the DAV triple combination significantly less effective. Therefore, it appears that to achieve maximal impact, all three components of the DAV triple combination of Doxycycline (1 μM), Azithromycin (1 μM) and Vitamin C (250 μM), should be administered at the same time.

Mechanistically, it appears that these pre-treatments “pre-conditioned MCF7 cells to the effects of the DAV triple combination. This may be due to their ability to induce oxidative stress, driving an anti-oxidant response.

Cancer: DAV combination therapy for targeting mitochondria in CSCs

Vitamin C is generally considered to be an anti-oxidant. However, depending on its relative concentration and cellular localization, Vitamin C can also act as a pro-oxidant, via the production of free radicals (). The ascorbate radical is normally very stable, but it becomes more reactive especially in the presence of metal ions, including iron (Fe), allowing the ascorbate radical to become a much more powerful pro-oxidant. Therefore, we speculate that as mitochondria are particularly rich in iron, they could become a key target of the pro-oxidant effects of Vitamin C, driving mitochondrial oxidative stress and new mitochondrial biogenesis.

Importantly, Vitamin C is highly concentrated within mitochondria [7–11]. For example, when U937 cells (a human leukaemia cell line) were incubated for only 15 minutes in media containing 3 μM Vitamin C, it was very efficiently transported to the mitochondria, reaching a level of 5 mM – being concentrated ~1,700-fold [8]. Mitochondrial transport of Vitamin C is accomplished by the sodium-coupled Vitamin C transporter 2 (SVCT2) [7–10], also known as SLC23A2, although other novel mitochondrial transporters have been suggested [11].

This mitochondrial targeting of Vitamin C may directly explain the effects we observed here of the triple combination on CSCs, as we have previously shown that CSCs have a significantly increased mitochondrial mass [12,13] and this contributes to their ability to undergo anchorage-independent growth [14,15]. Hence, the use of inhibitors of mitochondrial protein translation, together with Vitamin C, would ultimately prevent CSC mitochondria from fully recovering from the pro-oxidant effects of Vitamin C, as these target cells would be unable to re-synthesize new functional mitochondria (). Thus, under these metabolically restricted conditions, cancer cells would be expected to undergo “frustrated or “incomplete mitochondrial biogenesis. This assertion is directly supported by the Seahorse flux analysis data shown in , revealing i) reduced mitochondrial metabolism, ii) increased compensatory glycolytic function, and iii) severe ATP depletion.

Based on the mechanism(s) underpinning the strong effectiveness of the triple combination, other pro-oxidants could also be potentially substituted for Vitamin C. As many current chemotherapeutic agents, as well as targeted radiation, all kill cancer cells, via their pro-oxidant actions, then combined inhibition of mitochondrial protein translation could be used as an add-on to conventional therapy and would be predicted to improve their efficacy. However, Vitamin C clearly has fewer side effects and a better safety profile than most standard chemotherapeutic agents. Additional experimentation will be required to further test this intriguing hypothesis.

Previous studies have shown that Vitamin C by itself increases mitochondrial ATP production by up to 1.5-fold, in the rat heart, under conditions of hypoxia [16]. In addition, Vitamin C is a positive regulator of endogenous L-carnitine biosynthesis, an essential micro-nutrient that is required for mitochondrial beta-oxidation [17,18]. As such, these findings are consistent with our current results showing that Vitamin C alone is indeed sufficient to increase mitochondrial ATP production, by up to 2-fold, in MCF7 cells ().

Aging: Improving health-span and life-span

We believe that the DAV triple combination therapy that we describe here may also have implications for improving health-span and life-span, as aging is one of the most significant risk factors for the development of many human cancer types [19,20]. Moreover, we have previously demonstrated that Azithromycin, by itself, is an FDA-approved drug, with remarkable senolytic activity, that targets and removes senescent fibroblasts, such as myo-fibrobasts, with great efficiency approaching nearly 97% [21]. The accumulation of pro-inflammatory senescent cells is thought to be the primary cause of many aging-associated diseases, such as heart disease, diabetes, dementia and cancer, to name only a few [21]. Since cancer-associated fibroblasts (CAFs) are senescent myo-fibroblasts, with tumor promoting activity, this triple combination approach with Azithromycin may also effectively target the glycolytic tumor stroma of aggressive and metastatic cancers, especially those bearing the metabolic hallmarks of the “Reverse Warburg Effect [22–28].

Cell lines and reagents

MCF7 cells, an ER(+) human breast cancer cell line, was originally purchased from the American Type Culture Collection (ATCC), catalogue number HTB-22. Doxycycline, Azithromycin and Ascorbic Acid (Vitamin C) were all obtained commercially from Sigma-Aldrich, Inc.

Mammosphere formation assay

A single cell suspension was prepared using enzymatic (1x Trypsin-EDTA, Sigma Aldrich, #T3924), and manual disaggregation (25 gauge needle) [12–15]. Cells were plated at a density of 500 cells/cm² in mammosphere medium (DMEM-F12 + B27 + 20 ng/ml EGF + PenStrep) under non-adherent conditions, in culture dishes pre-coated with (2-hydroxyethylmethacrylate) (poly-HEMA, Sigma, #P3932), called “tumor-sphere plates. Vehicle alone (DMSO) control cells were processed in parallel. Cells were grown for 5 days and maintained in a humidified incubator at 37°C. After 5 days of culture, 3D mammospheres >50 μm were counted using an eye piece (“graticule), and the percentage of cells plated which formed spheres was calculated and is referred to as percent mammosphere formation efficiency (MFE) and was normalized to one (1 = 100% MFE).

Metabolic flux analysis

Real-time oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) rates in MCF7 cells were determined using the Seahorse Extracellular Flux (XFe96) analyzer (Seahorse Bioscience, USA) [29–31]. Briefly, 1.5 x 10⁴ cells per well were seeded into XFe96 well cell culture plates, and incubated overnight to allow cell attachment. Then, cells were treated with antibiotics and/or with Vitamin C for 72h. Vehicle-alone control cells were processed in parallel. After 72 hours of incubation, cells were washed in pre-warmed XF assay media (or for OCR measurement, XF assay media supplemented with 10mM glucose, 1mM Pyruvate, 2mM L-glutamine and adjusted at 7.4 pH). Cells were then maintained in 175 µL/well of XF assay media at 37°C, in a non-CO₂ incubator for 1 hour. During the incubation time, we loaded 25 µL of 80 mM glucose, 9 µM oligomycin, and 1M 2-deoxyglucose (for ECAR measurement) or 10 µM oligomycin, 9 µM FCCP, 10 µM rotenone, 10 µM antimycin A (for OCR measurement), in XF assay media into the injection ports in the XFe96 sensor cartridge. Measurements were normalized by protein content (SRB assay). Data sets were analyzed using XFe96 software and GraphPad Prism software, using one-way ANOVA and Student’s t-test calculations. All experiments were performed in quintuplicate, three times independently.

Live/dead assay for anoikis-resistance

Following monolayer treatment with either Doxycyline alone, Azithromycin alone or the combination for 48 hours, the CSC population was enriched by seeding onto low-attachment plates [32]. Under these conditions, the non-CSC population undergoes anoikis (a form of apoptosis induced by a lack of cell-substrate attachment) and CSCs are believed to survive. The surviving CSC fraction was then determined by FACS analysis. Briefly, 1 × 10⁴ MCF7 monolayer cells were treated with antibiotics or vehicle alone for 48h in 6-well plates. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 12h, the MCF7 cells were spun down. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Invitrogen) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). The live population was then identified by employing the LIVE/DEAD dye staining assay. Data were analyzed using FlowJo software.

We are grateful to Rumana Rafiq, for her kind and dedicated assistance, in keeping the Translational Medicine Laboratory at Salford running very smoothly. We would like to thank the Foxpoint Foundation and the Healthy Life Foundation for their philanthropic donations towards new equipment and infrastructure, in the Translational Medicine Laboratory at the University of Salford.

CONFLICTS OF INTEREST: MPL and FS hold a minority interest in Lunella Biotech, Inc.

FUNDING: This work was supported by research grant funding, provided by Lunella Biotech, Inc (to FS and MPL).