The thyroid hormone-αvß3 integrin axis in ovarian cancer: regulation of gene transcription and MAPK-dependent proliferation.

Ovarian carcinoma is the fifth common cause of cancer death in women, despite advanced therapeutic approaches. αvß3 integrin, a plasma membrane receptor, binds thyroid hormones (L-thyroxine, T4; 3,5,3'-triiodo-L-thyronine, T3) and is overexpressed in ovarian cancer. We have demonstrated selective binding of fluorescently labeled hormones to αvß3-positive ovarian cancer cells but not to integrin-negative cells. Physiologically relevant T3 (1 nM) and T4 (100 nM) concentrations in OVCAR-3 (high αvß3) and A2780 (low αvß3) cells promoted αv and ß3 transcription in association with basal integrin levels. This transcription was effectively blocked by RGD (Arg-Gly-Asp) peptide and neutralizing αvß3 antibodies, excluding T3-induced ß3 messenger RNA, suggesting subspecialization of T3 and T4 binding to the integrin receptor pocket. We have provided support for extracellular regulated kinase (ERK)-mediated transcriptional regulation of the αv monomer by T3 and of ß3 monomer by both hormones and documented a rapid (30-120 min) and dose-dependent (0.1-1000 nM) ERK activation. OVCAR-3 cells and αvß3-deficient HEK293 cells treated with αvß3 blockers confirmed the requirement for an intact thyroid hormone-integrin interaction in ERK activation. In addition, novel data indicated that T4, but not T3, controls integrin's outside-in signaling by phosphorylating tyrosine 759 in the ß3 subunit. Both hormones induced cell proliferation (cell counts), survival (Annexin-PI), viability (WST-1) and significantly reduced the expression of genes that inhibit cell cycle (p21, p16), promote mitochondrial apoptosis (Nix, PUMA) and tumor suppression (GDF-15, IGFBP-6), particularly in cells with high integrin expression. At last, we have confirmed that hypothyroid environment attenuated ovarian cancer growth using a novel experimental platform that exploited paired euthyroid and severe hypothyroid serum samples from human subjects. To conclude, our data define a critical role for thyroid hormones as potent αvß3-ligands, driving ovarian cancer cell proliferation and suggest that disruption of this axis may present a novel treatment strategy in this aggressive disease.

Tetraiodothyroacetic acid (tetrac) and nanoparticulate tetrac arrest growth of medullary carcinoma of the thyroid.

CONTEXT: Tetraiodothyroacetic acid (tetrac) blocks angiogenic and tumor cell proliferation actions of thyroid hormone initiated at the cell surface hormone receptor on integrin alphavbeta3. Tetrac also inhibits angiogenesis initiated by vascular endothelial growth factor and basic fibroblast growth factor. OBJECTIVE: We tested antiangiogenic and antiproliferative efficacy of tetrac and tetrac nanoparticles (tetrac NP) against human medullary thyroid carcinoma (h-MTC) implants in the chick chorioallantoic membrane (CAM) and h-MTC xenografts in the nude mouse. DESIGN: h-MTC cells were implanted in the CAM model (n = 8 per group); effects of tetrac and tetrac NP at 1 microg/CAM were determined on tumor angiogenesis and tumor growth after 8 d. h-MTC cells were also implanted sc in nude mice (n = 6 animals per group), and actions on established tumor growth of unmodified tetrac and tetrac NP ip were determined. RESULTS: In the CAM, tetrac and tetrac NP inhibited tumor growth and tumor-associated angiogenesis. In the nude mouse xenograft model, established 450-500 mm(3) h-MTC tumors were reduced in size over 21 d by both tetrac formulations to less than the initial cell mass (100 mm(3)). Tumor tissue hemoglobin content of xenografts decreased by 66% over the course of administration of each drug. RNA microarray and quantitative real-time PCR of tumor cell mRNAs revealed that both tetrac formulations significantly induced antiangiogenic thrombospondin 1 and apoptosis activator gene expression. CONCLUSIONS: Acting via a cell surface receptor, tetrac and tetrac NP inhibit growth of h-MTC cells and associated angiogenesis in CAM and mouse xenograft models.

Tetraidothyroacetic acid (tetrac) and tetrac nanoparticles inhibit growth of human renal cell carcinoma xenografts.

Renal cell carcinoma is the most lethal of the common urologic malignancies, with no available effective therapeutics. Tetrac (tetraiodothyroacetic acid) is a deaminated analogue of L-thyroxine (T(4)) that blocks the pro-angiogenesis actions of T(4) and 3, 5, 3'-triiodo-L-thyronine as well as other growth factors at the cell surface receptor for thyroid hormone on integrin alphavbeta3. Since this integrin is expressed on cancer cells and also on endothelial and vascular smooth cells, the possibility exists that Tetrac may act on both cell types to block the proliferative effects of thyroid hormone on tumor growth and tumor-related angiogenesis. To test this hypothesis, we determined the effect of Tetrac on tumor cell proliferation and on related angiogenesis of human renal cell carcinoma (RCC). We used two models: tumor cell implants in the chick chorioallantoic membrane (CAM) system and xenografts in nude mice. To determine the relative contribution of the nuclear versus the plasma membrane action of Tetrac, we compared the effects of unmodified Tetrac to Tetrac covalently linked to poly (lactide-co-glycolide) as a nanoparticle (Tetrac NP) that acts exclusively at the cell surface through the integrin receptor. In the CAM model, Tetrac and Tetrac NP (both at 1 microg/CAM) arrested tumor-related angiogenesis and tumor growth. In the mouse xenograft model, Tetrac and Tetrac NP promptly reduced tumor volume (p<0.01) when administered daily for up to 20 days. Animal weight gain was comparable in the control and treatment groups. Overall, the findings presented here provide evidence for the anti-angiogenic, and anti-tumor actions of Tetrac and Tetrac NP and suggest their potential utility in the treatment of renal cell carcinoma.

Spontaneous remission of cancer--a thyroid hormone dependent phenomenon?

Spontaneous remission (SR) of neoplasia is a rare biological event. Very few reports provide evidence for an eliciting event or mechanism. The only case in the literature of SR of lung cancer following myxedema coma is suggested to have been an instance of thyroid hormone deprivation-induced total tumor apoptosis. Review of the collective data suggests that the thyroid hormones modulate pleiotropic neoplasia--abetting mechanisms and that hypothyroidism may enhance the predisposition of neoplasms to spontaneous and therapy induced regression by lowering thresholds for apoptosis.

Antagonist effect of insulin-like growth factor I on protein kinase inhibitor-mediated apoptosis in human glioblastoma cells in association with bcl-2 and bcl-xL.

OBJECT: Tamoxifen (TAM) has been found to be effective in inhibiting proliferation of glioblastoma cells in vitro, but clinical studies have been disappointing. The purpose of this study was to determine whether insulin-like growth factor I (IGF-I), a potential autocrine/paracrine mitogen produced by glioblastomas, interferes with the antimitogenic actions of TAM. METHODS: Human glioblastoma cells were treated with or without TAM and/or IGF-I in vitro and evaluated for: viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide cleavage assay; apoptosis by histochemical analysis of nuclear morphology and 3'-OH DNA fragments; and expression of the IGF-I receptor, and the bcl-2, bcl-xL, and bax proteins by immunoblot analysis. In addition, p53 status was determined by DNA sequencing and by transient transfection with luciferase reporter plasmids containing wild-type or mutant p53. Results indicated that after 72 hours of exposure to 2 mg/ml TAM in vitro, 56.3% of WITG3 and 43.8% of U87-MG glioblastoma cells contained apoptotic nuclei (p < 0.01 compared with untreated cells). Apoptosis was independent of the presence of p53 because the WITG3 cells, in contrast to the U87-MG cells, expressed a mutant, nonfunctional p53. The WITG3 cells expressed IGF-I receptor proteins and demonstrated IGF-I binding. Exogenous IGF-I stimulated WITG3 cell proliferation and significantly (p < 0.05) antagonized the cytotoxic effects of TAM in a dose-dependent fashion; IGF-I, but not TAM, enhanced expression of bcl-2 and bcl-xL proteins; however, bax protein expression was unchanged by either treatment. CONCLUSIONS: Because many gliomas secrete large amounts of IGF-I in autocrine/paracrine growth pathways, these data may, in part, explain the failure of TAM to achieve clinical results as dramatic as those in vitro.

Thyroid hormone depletion inhibits astrocytoma proliferation via a p53-independent induction of p21 (WAF1/CIP1).

BACKGROUND: Clinical hypothyroidism has been associated with prolonged survival in several types of malignancies, but the mechanism of this effect is unknown. MATERIAL AND METHODS: In vitro studies of thyroid hormone depletion (via culture in medium containing 5% thyroid hormone-depleted fetal bovine serum (FBS)) were carried out using a human glioblastoma cell line (WITG3) which expresses a mutant, non-functional p53. RESULTS: Thyroid hormone depletion inhibited WITG3 proliferation compared to control medium containing 5% euthyroid FBS. There was no evidence of apoptosis and viability was not compromised. Cell cycle analysis by flow cytometry indicated that thyroid hormone depletion accumulated WITG3 cells in G1, with fewer cells progressing into S than in euthyroid medium. By immunoblotting, p21 (WAF1/CIP1) was only slightly detectable in lysates from WITG3 cells grown in control euthyroid medium; however, in thyroid hormone-depleted FBS, a marked induction of p2 1 occurred which could be reversed by exogenous thyroid hormone CONCLUSIONS: These data indicate that thyroid hormone depletion may cause a G, arrest in astrocytoma mediated by a p53-independent induction of p21 (WAF1/CIP1). Results suggest a mechanism which may explain the effect of hypothyroidism on suppression of tumor cell growth.

The thyroid gland as an intrinsic biologic response-modifier in advanced neoplasia--a novel paradigm.

The thyroid gland is the major endocrine modulator of physiological processes crucial to growth, maturation and metabolism. There is, however, a coherent body of evidence suggesting that the thyroid hormones modulate multiple neoplasia-dependent mechanisms. Recently spontaneous remission of metastatic non-small cell carcinoma of lung was reported in a man following recovery from myxedema coma. This rare biological event following a life-threatening clinical syndrome suggests that thyroid hormone deficiency directly or indirectly may significantly alter the balance between malignant tumor viability and growth on the one hand vs. cell death on the other. Evidence, therefore, is presented from clinical and experimental studies suggesting that decreased thyroid function (hypothyroidism) is associated with both enhanced response rates and unusual longevity. Possible mechanisms of action that may promote or retard neoplasia and are dependent on the functional state of the thyroid will be discussed. A novel paradigm is proposed; the thyroid gland aside from its known physiological activity is also the central modulator of solid neoplasia and therefore functions as an intrinsic biologic response-modifier of neoplasia. Induction of a clinically tolerable hypothyroid state in patients could become an integral part of the medical care of advanced cancer in conjunction with standard conventional modalities.

A prospective evaluation of thallium-201 single photon emission computerized tomography for brain tumor burden.

PURPOSE: The follow-up of patients with malignant brain tumors after surgery, radiation, and/or chemotherapy has been inadequate for evaluating brain tumor burden using computerized tomography (CT) or magnetic resonance imaging (MRI). Thallium-201 has been shown to concentrate in viable tumor, and Tl-201 single photon emission computerized tomography (SPECT) imaging can identify tumor burden more accurately than CT. METHODS AND MATERIALS: Thirty-one patients with glioblastoma and three patients with low grade astrocytoma were studied with Tl-201 SPECT. Histololgic diagnosis was obtained in all patients by biopsy and all patients had CT scans within 2 weeks of the SPECT study. Seventeen patients were followed with one or more SPECT and CT evaluations. RESULTS: Single photon emission computerized tomography studies, after surgery, radiotherapy, and/or chemotherapy, were more accurate than CT in identifying progression or regression of disease. Twenty-three patients had evidence of disease and 11 patients had no evidence of recurrent disease in the initial Tl-201 SPECT study following therapy. Computerized tomography identified 20 of the 23 patients with disease and 6 of 11 patients with no recurrent disease. Follow-up with Tl-201 SPECT in 17 patients suggested progression of disease in 9 patients, while CT showed progression in only 3 patients. Clinical examinations and repeat CT studies confirmed the accuracy of Tl-201 SPECT images. CONCLUSION: We found Tl-201 SPECT more accurate than CT scans in a prospective evaluation of 34 patients with brain tumor. Follow-up studies with both Tl-201 SPECT and CT imaging in 17 patients demonstrated that SPECT was more reliable than CT in identifying progression, improvement, or no change in brain tumor burden.

Selective enhancement of vincristine cytotoxicity in multidrug-resistant tumor cells by dilantin (phenytoin)

Tumor cell resistance to chemotherapeutic agents of diverse structure and mechanism of action is thought to be due to efflux of drug by P-glycoprotein, which is overexpressed in tumor cells with the multidrug-resistant phenotype. Agents generally associated with the multidrug-resistant phenotype include inhibitors of topoisomerase II, e.g., doxorubicin, etoposide, and the microtubule poisons such as vinblastine, vincristine (VCR), and taxol. The antiepileptic drug phenytoin (DPH), an inhibitor of tubulin polymerization, potentiates (P < 0.05) the cytotoxicity of the chemotherapeutically useful microtubule poison VCR in tumor cells with the wild-type or multidrug-resistant phenotype. Among agents associated with the multidrug-resistant phenotype, the modulation of cytotoxicity by DPH was selectively effective with the microtubule poison VCR but not the topoisomerase II inhibitor doxorubicin. The potentiation of vincristine cytotoxicity by DPH was not due to binding to P-glycoprotein or by increasing VCR accumulation. We thus propose a novel mechanism for the modulation of resistance based on evidence that DPH at noncytotoxic concentrations can selectively enhance the cytotoxic potential of vincristine without interfering with P-glycoprotein function. Thus, studies with phenytoin could assist in characterizing other molecular determinants of multidrug resistance and the design of trials to modulate drug efficacy.

Radiobiological studies of PC-3 and DU-145 human prostate cancer cells: x-ray sensitivity in vitro and hypoxic fractions of xenografted tumors in vivo.

PURPOSE: To determine the quantitative responses to x-irradiation of exponentially growing human prostatic cancer cell lines PC-3 and DU-145 in vitro, and to determine the hypoxic percentages of these two cell lines when grown in vivo as xenografted solid tumors in nude mice. METHODS AND MATERIALS: Radiation survival in vitro was quantitated using both single-hit, multitarget and linear-quadratic formalisms. Hypoxic fractions in vivo were determined from tumors of average sizes of about 750 mm3 using clonogenic excision assay. RESULTS: In vitro, the average single-hit, multitarget survival values for 7 replicate experiments for the DU-145 line were n = 1.92 (1.39-2.65), Dq(Gy) = 1.25, and Do(Gy) = 1.91 (1.88-1.94) (all values in parentheses indicate 95% confidence limits). For the PC-3 line (10 replicate experiments), these values were n = 2.84 (2.11-2.79), Dq(Gy) = 1.02, and Do(Gy) = 1.06 (0.87-1.25). For the linear-quadratic formalism, values of alpha(Gy-1 x 10(1) and beta(Gy-2 x 10(2) for the DU-145 and PC-3 lines were, respectively, 1.55 (0.42) and 5.21 (1.09); and 4.87 (1.11) and 5.50 (1.88). The mean percentage survival of the DU-145 and PC-3 lines at a dose of 2 Gy were, respectively, 59.8 (53.3-67.0) and 32.0 (25.8-38.2). In vivo, the hypoxic fractions for the DU-145 and PC-3 tumors were, respectively, 7.20 (4.30-11.5), and 52.3 (42.8-63.9). RESULTS: The data from the in vitro experiments show that the DU-145 cell line is significantly more radioresistant than the PC-3 cell line. In vivo, the DU-145 tumors exhibit a significantly lower hypoxic percentage than do PC-3 neoplasms. CONCLUSIONS: Results indicate that significantly variability exists within human prostate tumors in regard to both intrinsic radiosensitivity in vitro and levels of hypoxia in vivo. Because these data appear to be the first published information on the intrinsic radiosensitivity and intratumor hypoxia characteristics of human prostate cancer, additional studies are needed to define the distributional aspects of these clinically relevant endpoints.

Erythrocyte glutathione and tumour response to chemotherapy.

There is much evidence that tumour glutathione (GSH) concentration is an important factor in resistance to cancer chemotherapy. Since measurement of tumour GSH would require an invasive procedure in every patient, we have tried to find out whether GSH concentrations in peripheral-blood erythrocytes are related to the response to chemotherapy and thus whether they reflect those in tumour cells. Erythrocyte GSH concentrations were measured by spectrophotometry in peripheral blood from 28 patients with advanced breast cancer and 40 patients with other tumours before and after treatment with various conventional chemotherapeutic regimens. The mean pretreatment GSH concentration was lower in patients who showed a complete or partial response to chemotherapy than in those with stable or progressive disease in both the breast-cancer group (8.69 [95% confidence interval 5.99-11.39] vs 2.32 [1.23-3.41] mumol/g haemoglobin; p less than 0.01) and the group with other tumours (5.94 [4.14-7.74] vs 2.83 [1.71-3.95] mumol/g; p less than 0.01). The correlation of erythrocyte GSH concentration with response rate suggests that this measurement may be helpful in prediction of response to therapy.

Hypofractionated radiation therapy and concurrent cisplatin in malignant cerebral gliomas. Rapid palliation in low performance status patients.

Twenty-eight patients with high-grade cerebral gliomas (16 biopsy-proven and 12 diagnosed clinically and by computed tomography scan) were treated with altered fraction radiation and concomitant cisplatin (C-DDP). Twenty cases (Groups IA and IB) whose Karnofsky performance status (KPS) was 60% or less received hypofractionation and C-DDP. All these patients had received high-dose Decadron (Merck Sharp & Dohme, West Point, PA), and their conditions were not improving or progressively deteriorating. The first 11 patients (Group IA) received from 600 cGy twice weekly to 3600 cGy over 3 weeks combined with C-DDP IV at 40 mg/M2 every 2 weeks for two courses. The nine subsequent patients (Group IB) received from 600 cGy weekly to 3600 cGy over 5 to 6 weeks with C-DDP IV at 40 mg/M2 every 1 to 2 weeks for four courses. The target volume in all cases was confined to the tumor as defined on computed tomography (CT) scan with a 2 cm to 3 cm margin. The C-DDP at 40 mg/M2 was administered immediately (within 5 minutes after radiation). Eight cases (Group II) with a KPS of more than 60% were treated with hyperfractionation, i.e., from 200 cGy twice daily to 4800 cGy in just under 3 weeks. The C-DDP was administered every 2 weeks for a total of two courses, as for Group IA. In Group I, 15 of 20 (75%) patients experienced rapid improvement in their performance status, which usually becoming evident within 1 to 2 weeks from the initiation of treatment, and progressed over time. Four patients with a KPS of 10% improved their KPS to over 60%. This regimen was both well tolerated and logistically very convenient both for the patients and attending staff. Follow-up CT scans in three of 16 evaluable patients in the hypofractionated group showed complete tumor resolution. Median survival for Group IA was 7 months, for Group IB was 12 months, and overall was eight months. The Group II median survival was 9 months. This experience suggests that hypofractionated radiation in combination with C-DDP may offer rapid palliation with improvement in functional status in severely compromised patients with malignant glioma.