Somatostatin receptor subtype expression in human thyroid and thyroid carcinoma cell lines.

Somatostatin (SRIH) analogs can suppress the proliferation of human differentiated thyroid carcinoma cell lines that express SRIH receptors (SSTRs) demonstrated by radioligand binding analysis. Five distinct human SSTR subtypes (hSSTR1-5) that bind native SRIH exhibit diverse affinities to a wide range of SRIH analogs. Reverse transcriptase-PCR amplification of ribonucleic acids (RNAs) obtained from normal thyroid tissues and nine human thyroid carcinoma cell lines, grown as monolayer cultures and xenograft tumors in nude mice, were used to discriminate expression of SSTR subtype messenger RNAs (mRNAs). The cell lines were derived from a follicular adenoma (KAK-1), two follicular carcinomas (MRO-87 and WRO-82), two papillary carcinomas (NPA87 and KAT-10), and four anaplastic thyroid carcinomas (DRO-90, ARO-81, KAT-4, and KAT-18). Most thyroid cancer cell line monolayers and xenografts expressed SSTR3 and SSTR5 mRNAs. SSTR1 expression was more varied between monolayers and xenografts, whereas SSTR2 mRNA was only faintly detectable at the most extreme resolution. SSTR4 mRNA was faintly positive in only one anaplastic carcinoma xenograft. Normal thyroid also expressed SSTR3 and SSTR5 mRNAs, with only faint expression of SSTR1 and SSTR2 mRNAs (in one of five and three of five samples, respectively). SSTR mRNA expression was dependent upon in vitro culture conditions, as xenograft SSTR mRNA expression tended to decrease compared to that in each respective monolayer culture. Characterization of SSTR subtype expression in human thyroid carcinomas may permit targeting of specific SRIH analogs to inhibit proliferation of differentiated and anaplastic thyroid carcinomas in patients.

Multiple polyamine regulatory pathways control compensatory cardiovascular hypertrophy in coarctation hypertension.

While a number of factors may initiate structural alterations within the cardiovascular system in response to hypertension, there are obligate cellular signaling mechanisms, such as the polyamines, through which they must operate. This study examined the effects of polyamine synthesis inhibition using eflornithine, a suicide inhibitor of ornithine decarboxylase on blood pressure, compensatory remodeling of the cardiovascular system, and cardiac and aortic polyamine contents using an aortic coarctation model in rats. Eflornithine treatment failed to reduce carotid arterial blood pressure and actually significantly elevated vascular pressure above and below the coarctation site by 14 days of hypertension. Eflornithine only transiently reduced aortic polyamine content of hypertensive rats while this agent reduced coarctation-induced aortic medial wall thickening and the synthesis/deposition of fibronectin and laminin in the hypertensive aorta. Increases in left ventricular mass and polyamine content were concomitantly reduced in hypertensive rats administered eflornithine. These results suggest that multiple polyamine regulatory pathways may maintain vascular polyamine content in response to aortic coarctation; however de novo polyamine synthesis is essential for select aspects of vascular remodeling, including matrix synthesis. Cardiac tissue, in contrast, may rely principally on de novo polyamine synthesis.

Eflornithine alters changes in vascular responsiveness associated with coarctation hypertension.

This study examined the temporal effects of the polyamine synthesis inhibitor eflornithine (alpha-difluoromethylornithine) on vascular responses to KCI, norepinephrine, sodium nitroprusside and acetylcholine in aortic rings from coarctation hypertensive rats. Coarctation hypertension reduced the contractile response of aortic rings to KCI and norepinephrine, increased sensitivity (reduced the EC50 value) to norepinephrine and attenuated relaxation to acetylcholine by 14 days of hypertension. Treatment of coarctation hypertensive rats with eflornithine resulted in a normalization of the contractile intensity to KCI and norepinephrine and relaxations to acetylcholine by 14 days of hypertension. Responses to sodium nitroprusside were similar in all groups at all time points. Hyperresponsiveness to norepinephrine produced by coarctation of the aorta was not affected by eflornithine. These studies indicate that normalization of vascular function can occur in the presence of significantly elevated blood pressure upon chronic administration of eflornithine. This functional normalization correlates with eflornithine-mediated regression of structural abnormalities normally associated with pressure overload hypertension.

Antineoplastic activity of taxol against human anaplastic thyroid carcinoma cell lines in vitro and in vivo.

Anaplastic thyroid carcinoma is a rapidly fatal neoplasm that fails to adequately respond to any known chemotherapeutic regimen. We tested taxol (paclitaxel) against six human anaplastic thyroid carcinoma cell lines (DRO-90, ARO-81, KAT-4, KAT-18, SW-1736, and BHT-101). Each cell line monolayer culture, in log phase growth, was treated with taxol concentrations ranging from 0.001-5.0 mumol/L. Cell numbers, after 24-, 48-, and 72-h growth periods in separate experiments, were expressed as percentages of control cell numbers without taxol. All cell lines showed maximal inhibition with 0.05 mumol/L taxol at 3-28% of control cell numbers. Greater inhibition was seen with longer growth periods. Three cell lines (DRO-90, ARO-81, and KAT-4) were grown as sc xenograft tumors in nude mice for 18-26 days. Treatment groups received sc taxol injections in sites distant from the tumors, whereas control mice received vehicle. All taxol-treated xenografts were inhibited to near-starting volume or disappeared, whereas control xenograft volumes increased 9- to 59-fold. These results suggest that taxol may have beneficial clinical effects in anaplastic thyroid carcinoma patients.

The potential of a novel polyamine transport inhibitor in cancer chemotherapy.

The polyamines, putrescine (PUT), spermidine (SPD) and spermine (SPM), are a family of low molecular weight organic cations that are essential for cell growth, differentiation and neoplastic transformation. The marked compensatory increase in extracellular polyamine influx may be a reason for the unsatisfactory clinical chemotherapeutic effect of polyamine synthesis blockers like difluoromethylornithine (DFMO). In this study, a polymeric conjugate of SPM (poly-SPM) that blocks the import of polyamines into mammalian cells was used to test the potential therapeutic exploitation of the polyamine transport system in anticancer therapy. Our results indicate that a temperature-dependent polyamine transport system is expressed in two human cancer cell lines, MES-SA uterine sarcoma cells, K562 leukemic cells and their respective multiple drug resistance (MDR) positive counterparts, Dx5 and K562/R7 cells. The V(max) values for 14C-PUT and 14C-SPD uptake were significantly higher in MES-SA than in Dx5 cells, whereas the respective Km values were significantly lower. Addition of 20 microM poly-SPM reduced both the uptake of 14C-polyamines and the cellular polyamine contents in both cancer cell lines. In addition, the poly-SPM conjugate evoked a concentration-dependent cytotoxicity in MES-SA and K562 cells and their MDR-positive variants. Presence of aminoguanidine, an amine oxidase blocker, failed to alter the IC50 values generated with poly-SPM, which indicates that this polymer is not a substrate for amine oxidase. Moreover, coadministration of 25 microM SPD reversed the cytotoxic effect exerted by poly-SPM on both the MES-SA and Dx5 cells as reflected by an increase in their IC50 values. Relative to parental cells, the MDR-positive variants exhibited a lower 14C-polyamine uptake rate and were more resistant to the cytotoxic effect of poly-SPM. Pretreatment with 1 mM DFMO for 24 hr significantly increased polyamine transport, but failed to reduce intracellular SPM contents or exert a cytotoxic effect in both cancer cell lines. On other hand, the combination of DFMO and poly-SPM produced a greater depletion of polyamine content accompanied by a higher cytotoxicity than either agent alone. These results provide the first direct evidence that pharmacologic interruption of polyamine uptake may be an effective approach to cancer therapy. In addition, it appears that expression of MDR influences polyamine transport and renders cells more resistant to the cytotoxic effects of SPM polymer.

Reversal of doxorubicin, etoposide, vinblastine, and taxol resistance in multidrug resistant human sarcoma cells by a polymer of spermine.

We have previously described the synthesis of a cytotoxic polymeric conjugate of spermine (Poly-SPM) which is able to inhibit the transport of polyamines (spermine, spermidine, and putrescine) into normal and malignant cells. Recent studies examining the toxicity of Poly-SPM in parental and multidrug resistant (MDR) cancer cells have revealed a cross-resistance in the MDR variant Dx5 to the toxic effects of the conjugate in the MDR-positive cells. There were also differences in spermine and putrescine uptake rates between parental and MDR-positive with the MDR-positive cells having a lower Vmax and a higher Km. The ability of this Poly-SPM to reverse MDR was examined in MDR variants (Dx5 cells) of the human sarcoma cell line MES-SA. The cells express high levels of the mdr1 gene product, P-glycoprotein, and are 25-to 60-fold resistant to doxorubicin (DOX), etoposide (VP-16), vinblastine (VBL), and taxol (TAX). Cytotoxicity was measured by the MTT [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Poly-SPM (50 microM) lowered the drug concentration IC50 values in the Dx5 cells by 37-fold with VBL, 42-fold with DOX, 29-fold with VP-16, and 25-fold with TAX when compared to the control IC50 values without Poly-SPM. This reversal of resistance was concentration dependent, decreasing 17-fold with DOX, 6.1-fold with VBL, 19-fold with VP-16, and 5-fold with TAX when 25 microM Poly-SPM was used. No modulation was observed in the parental cell line MES-SA, which does not express the mdr1 gene. Poly-SPM had no influence on the IC50 of non-MDR chemotherapeutic agents such as cisplatin. The modulation studies correlated with the ability of Poly-SPM to reverse the cellular accumulation defect of [3H]-VBL and [3H]-TAX in the Dx5 but not MES-SA cells. Pretreatment of the Dx5 cell with alpha-difluoromethylornithine (DFMO at 2 and 5 microM) for 24 h increased the function of the MDR transporter to further decrease the cellular accumulation of VBL and TAX when compared to untreated cells. DFMO pretreatment is known to upregulate the polyamine transporter(s). These findings show that, in addition to inhibiting polyamine transport, Poly-SPM reverses MDR in Dx5 cells, suggesting a potential relationship between the polyamine influx transporter and the MDR efflux pump. This potential functional link between the polyamine influx transporter(s) and the MDR efflux transporter (P-glycoprotein) offers a novel approach to inhibiting this form of drug resistance.