Mitochondria as oncotarget: a comparison between the tetracycline analogs doxycycline and COL-3

Abstract

Tetracyclines have anticancer properties in addition to their well-known antibacterial properties. It has been proposed that tetracyclines slow metastasis and angiogenesis through inhibition of matrix metalloproteinases. However, we believe that the anticancer effect of tetracyclines is due to their inhibition of mitochondrial protein synthesis, resulting in a decrease of the mitochondrial energy generating capacity. Several groups have developed analogs that are void of antibacterial action. An example is COL-3, which is currently tested for its anticancer effects in clinical trials. We have undertaken a comparative study of the tetracycline analogs COL-3 and doxycycline, which has an antibacterial function, to further investigate the role of the mitochondrial energy generating capacity in the anticancer mechanism and, thereby, evaluate the usefulness of mitochondria as an oncotarget. Our experiments with cultures of the human A549, COLO357 and HT29 cancer cells and fibroblasts indicated that COL-3 is significantly more cytotoxic than doxycycline. Mitochondrial translation assays demonstrated that COL-3 has retained its inhibitory effect on mitochondrial protein synthesis. Both drugs caused a severe decrease in the levels of mitochondrially encoded cytochrome-c oxidase subunits and cytochrome-c oxidase activity. In addition, COL-3 produced a marked drop in the level of nuclear-encoded succinate dehydrogenase subunit A and citrate synthase activity, indicating that COL-3 has multiple inhibitory effects. Contrary to COL-3, the anticancer action of doxycycline appears to be based specifically on inhibition of mitochondrial protein synthesis, which is thought to affect rapidly proliferating cancer cells more than healthy tissue. Doxycycline is likely to cause less side effects that COL-3.

Mitochondria as target to inhibit proliferation and induce apoptosis of cancer cells: the effects of doxycycline and gemcitabine

“…Several groups have reported that doxycycline induces apoptosis in other cancer cell lines at higher doxycycline concentrations than used here 24,49,50,55 . We found that doxycycline treatment did not induce caspase 9, 8 or 3/7 activity in A549 cells, supporting our earlier observations that doxycycline does not cause activation of procaspase 3 52 . However, doxycycline and gemcitabine co-treatment resulted in a strong induction of caspase 9 and 3/7 activity when compared to gemcitabine alone.…”

“…The western blots showed that the doxycycline-induced decrease of MTCO1 and MTCO2 in A549 cells flattens after 3 days of treatment. Earlier, we showed that a proliferation slowdown of A549 cells occurs after 3 days of doxycycline treatment 52 . Apparently, at this point, the mitochondrial energy generating capacity is too low to maintain normal cell proliferation.…”

“…After 4 days of treatment, A549 and A549 ρ° cells were replated at ~50% confluency in wells of a 6-well tissue culture plate and treatment was continued. The next day, when ~85% confluency was reached, mitochondrial translation products were labelled with l-[ 35 S]-methionine (Perkin Elmer), followed by protein extraction, resolution on polyacrylamide gels, Coomassie staining and autoradiography as described 52 . Images of the stained gels and autorads were captured with the BioRad Chemidoc MP Imaging System.…”

“…We found that the synthesis of 11 of the 13 mitochondrial translation products was inhibited by doxycycline, while the synthesis of two mitochondrial translation products, MTATP6 and MTATP8, was increased. We do not know the mechanism producing this increase but we suspect that it involves a response to the long-term exposure of doxycycline because in earlier experiments, in which the cells were exposed to doxycycline for 2 hours, this increase was not noted 52 . The inhibition of the synthesis of the 11 mitochondrial translation products was modest but dose-response experiments by others have indicated that the 10 μg/ml of doxycycline we used results in maximum inhibition 51 .…”

“…To assess the effect of doxycycline on mitochondrial protein synthesis, we labelled the mitochondrial translation products with [ 35 S]-methionine, followed by separation on gels and autoradiography. Others have used radioactive assays followed by scintillation counting to measure doxycycline-mediated inhibition of mitochondrial protein synthesis 15,51 but, to our knowledge, autoradiographs with samples from doxycycline-treated cells showing the individual mitochondrial translation products have not been documented earlier, except in our recent publication 52 . We found that the synthesis of 11 of the 13 mitochondrial translation products was inhibited by doxycycline, while the synthesis of two mitochondrial translation products, MTATP6 and MTATP8, was increased.…”