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

Doxycycline has anti-tumour effects in a range of tumour systems. The aims of this study were to define the role mitochondria play in this process and examine the potential of doxycycline in combination with gemcitabine. We studied the adenocarcinoma cell line A549, its mitochondrial DNA-less derivative A549 ρ° and cultured fibroblasts. Treatment with doxycycline for 5 days resulted in a decrease of mitochondrial-encoded proteins, respiration and membrane potential, and an increase of reactive oxygen species in A549 cells and fibroblasts, but fibroblasts were less affected. Doxycycline slowed proliferation of A549 cells by 35%. Cellular ATP levels did not change. Doxycycline alone had no effect on apoptosis; however, in combination with gemcitabine given during the last 2 days of treatment, doxycycline increased caspase 9 and 3/7 activities, resulting in a further decrease of surviving A549 cells by 59% and of fibroblasts by 24% compared to gemcitabine treatment alone. A549 ρ° cells were not affected by doxycycline. Key effects of doxycycline observed in A549 cells, such as the decrease of mitochondrial-encoded proteins and surviving cells were also seen in the cancer cell lines COLO357 and HT29. Our results suggest that doxycycline suppresses cancer cell proliferation and primes cells for apoptosis by gemcitabine.

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

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.

Clonal expansion of T cells in abdominal aortic aneurysm: a role for doxycycline as drug of choice?

Most reported studies with animal models of abdominal aortic aneurysm (AAA) and several studies with patients have suggested that doxycycline favourably modifies AAA; however, a recent large long-term clinical trial found that doxycycline did not limit aneurysm growth. Thus, there is currently no convincing evidence that doxycycline reduces AAA expansion. Here, we critically review the available experimental and clinical information about the effects of doxycycline when used as a pharmacological treatment for AAA. The view that AAA can be considered an autoimmune disease and the observation that AAA tissue shows clonal expansion of T cells is placed in the light of the well-known inhibition of mitochondrial protein synthesis by doxycycline. In T cell leukaemia animal models, this inhibitory effect of the antibiotic has been shown to impede T cell proliferation, resulting in complete tumour eradication. We suggest that the available evidence of doxycycline action on AAA is erroneously ascribed to its inhibition of matrix metalloproteinases (MMPs) by competitive binding of the zinc ion co-factor. Although competitive binding may explain the inhibition of proteolytic activity, it does not explain the observed decreases of MMP mRNA levels. We propose that the observed effects of doxycycline are secondary to inhibition of mitochondrial protein synthesis. Provided that serum doxycycline levels are kept at adequate levels, the inhibition will result in a proliferation arrest, especially of clonally expanding T cells. This, in turn, leads to the decrease of proinflammatory cytokines that are normally generated by these cells. The drastic change in cell type composition may explain the changes in MMP mRNA and protein levels in the tissue samples.

Regulatory aspects of allergen products for immunotherapy--considerations from the viewpoint of manufacturers.

The present publication describes the actual situation anno 2005 with respect to registration of allergen products for specific immunotherapy (SIT) in Europe. It is concluded that the lack of the implementation of regulations and directives in force, is due to underestimation of the specific demands and aspects of the pathogenicity of allergic diseases at the one hand and of allergen products as a unique set of biological medicinal products at the other. The issues of regulatory nature that need careful attention and consideration in the opinion of the manufacturers are given in a number of statements. The main conclusion is that an EU-guideline for allergen products is urgently needed. A dialogue between regulatory authorities and representatives of the manufacturers, prior to the formulation of a new guideline seems of the utmost importance, as to prevent a similar stalemate as after the introduction of the foreseen regulatory measures of the past. The new guideline should include: An allergen product monograph. Recommendations for clinical development of allergen products like: Number of patients for efficacy and safety Acceptability of different efficacy parameters. Recommendations for toxicology.

The EAMG position on the regulation of existing products for treatment with special reference to named patient products (NPPs).

Allergen products have a long history in both diagnosis and management of allergic disease. It is emphasized that the availability of named patient products presents a valuable and indispensable option for the effective and safe treatment of patients suffering from IgE-mediated allergic diseases. A regulatory climate should be achieved in which these products can survive until a safe and effective alternative is borne out. Within the context of the paper the following definition is used: A named patient product or NPP is an allergen product, prepared with a specific reference number in accordance with a prescription for an individual patient, identified by the name of this patient and the reference number, and delivered after control of consistency with previous treatments. It is stressed that the standards of quality that hold for registered products are also applicable for named patient preparations which, for various reasons, can not be filed for registration. The paper frequently refers to a Position Paper that has recently been adopted by the European Allergen Manufacturers Group, the EAMG. The presentation breaks a lance for a concerted approach of the NPP-concept by striving at harmonization of legal, regulatory, manufacturing, and distribution conditions.