Activation of tumor cell integrin αvß3 by radiation and reversal of activation by chemically modified tetraiodothyroacetic acid (tetrac).

PURPOSE: Integrin αvß3 is an important structural and signaling protein of the plasma membrane of cancer cells and dividing blood vessel cells. The plastic extracellular domain of the protein binds to extracellular matrix proteins and plasma membrane proteins, changing cell-cell interactions and generating intracellular signals that influence cell behavior. αvß3 also contains a receptor for thyroid hormone and derivatives, including tetraiodothyroacetic acid (tetrac). MATERIALS AND METHODS: Human prostate cancer (PC3) cells were engrafted in the chicken chorioallantoic membrane model. The well-vascularized spheroidal xenografts were exposed to X-radiation in varying dosages (1-10 Gy) and in the presence and absence of an antibody that recognizes unliganded human ß3 integrin monomer in the extended or open (activated) configuration. RESULTS: Radiation significantly increased activated ß3 within 1 h (P < .001), a radiation response not previously reported. Incubation of cells with unmodified tetrac or tetrac covalently linked to a nanoparticle (Nanotetrac, NDAT) did not change basal activation state of the integrin monomer, but prevented radiation-induced activation of ß3. CONCLUSIONS: Activation of the integrin in response to radiation is interpreted as a defensive response, perhaps leading to increased intercellular affinity and inhibition of cell division, a radioresistant state. Action of NDAT indicates that pharmacologic interventions in the radiation response of integrin ß3 monomer and therefore of αvß3 are feasible.

Radioresistance of cancer cells, integrin αvß3 and thyroid hormone.

Radioresistance is a substantial barrier to success in cancer management. A number of molecular mechanisms support radioresistance. We have shown experimentally that the thyroid hormone analogue receptor on the extracellular domain of integrin αvß3 may modulate the state of radiosensitivity of tumor cells. Specifically, tetraiodothyroacetic acid (tetrac), a derivative of L-thyroxine (T4), can reduce radioresistance in cancer cells. In this review, we list a number of intrinsic signal transduction molecules and other host factors that have been reported to support/induce radioresistance in cancer cells and that are also subject to control by T4 through actions primarily initiated at integrin αvß3. Additional preclinical evidence is needed to support these radioresistance-relevant actions of thyroid hormone.

In vitro effects of tetraiodothyroacetic acid combined with X-irradiation on basal cell carcinoma cells.

We investigated radiosensitization in an untreated basal cell carcinoma (TE.354.T) cell line and post-pretreatment with tetraiodothyroacetic acid (tetrac) X 1 h at 37°C, 0.2 and 2.0 µM tetrac. Radioresistant TE.354.T cells were grown in modified medium containing fibroblast growth factor-2, stem cell factor-1 and a reduced calcium level. We also added reproductively inactivated (30 Gy) "feeder cells" to the medium. The in vitro doubling time was 34.1 h, and the colony forming efficiency was 5.09 percent. These results were therefore suitable for clonogenic radiation survival assessment. The 250 kVp X-ray survival curve of control TE.354.T cells showed linear-quadratic survival parameters of αX-ray = 0.201 Gy-1 and ßX-ray = 0.125 Gy-2. Tetrac concentrations of either 0.2 or 2.0 µM produced αX-ray and ßX-ray parameters of 2.010 and 0.282 Gy-1 and 2.050 and 0.837 Gy-2, respectively. The surviving fraction at 2 Gy (SF2) for control cells was 0.581, while values for 0.2 and 2.0 µM tetrac were 0.281 and 0.024. The SF2 data show that tetrac concentrations of 0.2 and 2.0 µM sensitize otherwise radioresistant TE.354.T cells by factors of 2.1 and 24.0, respectively. Thus, radioresistant basal cell carcinoma cells may be radiosensitized pharmacologically by exposure to tetrac.

Corrigendum: "Cancer Cell Gene Expression Modulated from Plasma Membrane Integrin αvß3 by Thyroid Hormone and Nanoparticulate Tetrac".

[This corrects the article on p. 240 in vol. 5, PMID: 25628605.].

Nanotetrac targets integrin αvß3 on tumor cells to disorder cell defense pathways and block angiogenesis.

The extracellular domain of integrin αvß3 contains a receptor for thyroid hormone and hormone analogs. The integrin is amply expressed by tumor cells and dividing blood vessel cells. The proangiogenic properties of thyroid hormone and the capacity of the hormone to promote cancer cell proliferation are functions regulated nongenomically by the hormone receptor on αvß3. An L-thyroxine (T4) analog, tetraiodothyroacetic acid (tetrac), blocks binding of T4 and 3,5,3'-triiodo-L-thyronine (T3) by αvß3 and inhibits angiogenic activity of thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvß3 beyond the inhibition of binding of T4 and T3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvß3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies.

Cancer Cell Gene Expression Modulated from Plasma Membrane Integrin αvß3 by Thyroid Hormone and Nanoparticulate Tetrac.

Integrin αvß3 is generously expressed by cancer cells and rapidly dividing endothelial cells. The principal ligands of the integrin are extracellular matrix proteins, but we have described a cell surface small molecule receptor on αvß3 that specifically binds thyroid hormone and thyroid hormone analogs. From this receptor, thyroid hormone (l-thyroxine, T4; 3,5,3'-triiodo-l-thyronine, T3) and tetraiodothyroacetic acid (tetrac) regulate expression of specific genes by a mechanism that is initiated non-genomically. At the integrin, T4 and T3 at physiological concentrations are pro-angiogenic by multiple mechanisms that include gene expression, and T4 supports tumor cell proliferation. Tetrac blocks the transcriptional activities directed by T4 and T3 at αvß3, but, independently of T4 and T3, tetrac modulates transcription of cancer cell genes that are important to cell survival pathways, control of the cell cycle, angiogenesis, apoptosis, cell export of chemotherapeutic agents, and repair of double-strand DNA breaks. We have covalently bound tetrac to a 200 nm biodegradable nanoparticle that prohibits cell entry of tetrac and limits its action to the hormone receptor on the extracellular domain of plasma membrane αvß3. This reformulation has greater potency than unmodified tetrac at the integrin and affects a broader range of cancer-relevant genes. In addition to these actions on intra-cellular kinase-mediated regulation of gene expression, hormone analogs at αvß3 have additional effects on intra-cellular protein-trafficking (cytosol compartment to nucleus), nucleoprotein phosphorylation, and generation of nuclear coactivator complexes that are relevant to traditional genomic actions of T3. Thus, previously unrecognized cell surface-initiated actions of thyroid hormone and tetrac formulations at αvß3 offer opportunities to regulate angiogenesis and multiple aspects of cancer cell behavior.

Radiosensitization and production of DNA double-strand breaks in U87MG brain tumor cells induced by tetraiodothyroacetic acid (tetrac).

We describe the steady-state levels and molecular and cellular repair of DNA double-strand breaks (DSBs) in tetraiodothyroacetic acid (tetrac)-treated human U87MG glioblastoma cells after x-irradiation in vitro. This study was conducted to provide a basis for our previous observation of radiosensitization and inhibition of cellular recovery after irradiation of tetrac-exposed GL261 murine brain tumor cells. We used the neutral comet assay to assess DSBs, and found that the steady-state DSB levels as indicated by the mean tail moment after a 1 h application of 2 nM tetrac at 37 °C was increased from a value of 6.1 in control cells to 12.4 in tetrac treated cells at 0 radiation dose. However, at all radiation doses, the induction curves of DSBs were parallel, suggesting that no interaction of tetrac with the initial physical-chemical actions of ionizing radiation occurred. Flow cytometric measurements indicated that this increase was not due to alterations in the relative percentages of U87MG cells throughout the cell cycle. In split-dose DNA repair studies we found that tetrac decreased the repair rate of U87 cells by a factor of 72.5%. This suggests that the radiosensitization from graded single doses of x-rays occurs as a consequence of tetrac inhibition of the post-irradiation repair process. These results link the previously noted changes in cellular endpoints to a molecular endpoint. That is, tetrac produces increased numbers of DSBs in the unirradiated steady-state coupled with a decreased repair rate of DSBs in fractionated radiation experiments.

Radiosensitization of GL261 glioma cells by tetraiodothyroacetic acid (tetrac).

We studied effects of tetrac (tetraiodothyroacetic acid) on survival of GL261, a murine brain tumor cell line, following single doses of 250 kVp x-rays and on repair of damage (sublethal and potentially lethal damage repair; SLDR, PLDR) in both exponential and plateau phase cells. Cells were exposed to 2 muM tetrac (1 h at 37 degrees C) prior to x-irradiation. At varying times after irradiation, cells were re-plated in medium without tetrac. Two weeks later, colonies were counted and results analyzed using either the linear-quadratic (LQ) or single-hit, multitarget (SHMT) formalisms. Tetrac sensitized both exponential and plateau phase cells to x-irradiation, as shown by a decrease in the quasi-threshold dose (Dq), leading to an average tetrac enhancement factor (ratio of SF2 values) of 2.5. Tetrac reduced SLDR in exponential cells by a factor of 1.8. In plateau phase cells there was little expression of SLDR, but tetrac produced additional cell killing at 1-4 h after the first dose. For PLDR expression in exponential cells, tetrac inhibited PLDR by a factor of 1.9, and in plateau phase cells, tetrac decreased PLDR expression by a factor of 3.4. These data show that the decreased Dq value seen after single doses of x-rays with tetrac treatment is also accompanied by a significant decrease in recovery from sublethal and potentially lethal damage.

Enhancement of radiosensitivity of the MCF-7 breast cancer cell line with human chorionic gonadotropin.

Secretion of human chorionic gonadotropin (hCG) during pregnancy induces differentiation of the mammary gland, thereby making breast tissue less susceptible to carcinogenesis. HCG binds to specific hCG receptors on mammary epithelial cells inducing changes in gene expression that can inhibit cell proliferation and, therefore, interfere with tumorigenesis. Since breast cancer cells also contain a relatively high level of the hCG receptor, hCG has potential as a therapeutic agent. We postulated that hCG might also enhance the radiosensitivity of breast cancer cells and, therefore, be useful as an adjunctive therapy. In the present study, MCF-7 breast cancer cells grown in cell culture were treated with hCG (0.2-5 IU/ml) for 24 h prior to exposing the cells to 0 Gy, 3 Gy, 4 Gy, or 5 Gy of radiation. Following irradiation, the MCF-7 cells were incubated either in the presence or absence of hCG. Cell survival was monitored with an MTT assay 1 day, 4 days, and 7 days after irradiation. All of the concentrations of hCG tested enhanced radiosensitivity of MCF-7 cells. The maximum enhancement occurred with MCF-7 cells that had been exposed to 2 IU/ml of hCG for at least 24 h prior to irradiation with 4 Gy. The use of higher concentrations of hCG or a higher dose of radiation did not increase the enhancement effect. Treatment of MCF-7 cells with hCG for only 24 h was sufficient to achieve the maximum effect. However, maintaining the cells in hCG beyond 24 h increased the effectiveness of the lowest hCG concentration. Using a linear-quadratic equation to analyze the data, we determined that the use of hCG would result in an 8-10% reduction in MCF-7 cell survival at a dose of 2 Gy, a typical dose used in conventional cancer therapy.