L-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines.

L-Canavanine (L-CAV) is a naturally occurring L-arginine analog that induces the formation of non-functional proteins in a variety of organisms. Previous studies have shown that L-CAV is cytotoxic for several human tumor cell lines. In this study, we have evaluated the cytotoxicity of L-CAV for both parental and multi-drug resistant (MDR) human tumor cells. We have also determined the effect of L-CAV exposure on cellular expression and activity of the MDR P-glycoprotein (P-gp) membrane efflux pump, and the effect of L-CAV on cellular accumulation of P-gp substrates. The effect of pre-treatment with non-cytotoxic doses of L-CAV on cellular sensitivity to ten standard antineoplastic agents was also evaluated, in order to assess the chemosensitization potential of L-CAV. 3-(4,5-Dimethylthiazol-)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assays revealed that the MDR variants of human uterine sarcoma and leukemic cells were equally sensitive to L-CAV as compared with their respective parental controls. Although the presence of free L-CAV in the uptake media did not influence cellular accumulation of P-gp substrates, cells cultured for 72 h in 250 microM L-CAV accumulated from 16 to 23% less P-gp substrate than untreated controls. Although L-CAV-cultured sarcoma cells accumulated 17% less doxorubicin (DOX) than untreated controls, they were three times more sensitive to its cytotoxic effects. L-CAV-treated cells were also significantly more sensitive to cisplatin, 5-fluorouracil, mitoxantrone and bleomycin than were untreated controls. Indirect immunofluorescence revealed that 72-h exposure to as much as 1000 microM L-CAV did not alter cellular expression of P-gp. These studies suggest that L-CAV may be equally cytotoxic for both parental and MDR tumor cells, and that L-CAV neither induces the expression of, nor is a substrate for, P-gp. The observation that L-CAV pre-treatment reduces cellular accumulation of DOX, yet sensitizes tumor cells to DOX and other DNA-targeting antineoplastic drugs, suggests a role for L-CAV as a chemosensitizer for the chemotherapy of cancer.

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.