Human Uterine Sarcoma Cell Line Research Articles

Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5

The uterine sarcoma human cell line MES-SA/Dx5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 150 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recognized by the lectins in the digoxigenin (DIG) glycan differentiation kit from Roche Allied Science, all of which were N-glycans. Pgp was isolated using both slab and preparative gel elution. The monoclonal antibody C219 was used to identify the presence of Pgp and Pgp treated with PNGase F on our blots. Pgp isolated from MES-SA/Dx5 cells contains at least two different complex N-glycans — one high mannose tree, detected by GNA, and one branched hybrid oligosaccharide-capped with terminal sialic acids, detected by SNA and MAA. DSA, specific for biantennary oligosaccharides possessing β(1–4)- N-acetyl- d-glucosamine residues, also recognized the blotted Pgp and is probably detecting the core Galβ(1–4)–GlcNAc x component found in other Pgps.

Antitumor effect of TNP‐470, an angiogenesis inhibitor, combined with ultrasound irradiation for human uterine sarcoma xenografts evaluated using contrast color Doppler ultrasound

Microvascular endothelial cells, which are recruited by tumors, have become an important target in cancer therapy. This study firstly examined the antitumor effect of angiogenesis inhibitor combined with ultrasound (US) irradiation for human cancer in vivo and evaluated its vascularity using color Doppler US in real time with a microbubble US contrast agent. A human uterine sarcoma cell line, FU-MMT-1, was used in vivo because this tumor is one of the most malignant neoplasms of the human solid tumors and it also has a poor response to any of the chemotherapeutic agents currently used, as well as to radiotherapy. In angiogenic inhibitors, TNP-470 was selected to use in an in vivo study, because this agent showed a higher inhibitory effect in tube formation assay in vitro, than that of FR118487, or thalidomide. The FU-MMT-1 xenografts in nude mice were treated using US at a low-intensity (2.0 w/cm(2), 1MHZ) for 4 min three times per week each after the subcutaneous injection of TNP-470 (30 mg/kg), an angiogenesis inhibitor, and this treatment was continued for 8 weeks. Either treatment of US alone or TNP-470 alone showed a suppression of tumor growth, in comparison to the non-treatment group (control), and a significantly enhanced effect was obtained using the combined treatment. A reduction in the intratumoral vascularity, which was evaluated using both color Doppler and immunohistochemistry, was significantly demonstrated using the combined treatment, in comparison to each treatment alone, and the control. No side-effect was observed in any mice in the combined treatment group. These results suggest that the antitumor effect of TNP-470 for uterine sarcoma was accelerated by US irradiation in vivo and this combination might be a potentially effective for new cancer therapy.

The effects of the CDK inhibitor seliciclib alone or in combination with cisplatin in human uterine sarcoma cell lines

Objectives. Inhibition of cyclin-dependent-kinases (CDKs) represents an interesting approach in cancer therapy. We have explored this in cell lines of human uterine sarcoma-tumours associated with poor survival, chemo-unresponsiveness and deregulation of cell cycle components. We studied the effects of the CDK inhibitor seliciclib (CYC202, R-roscovitine) when used alone or in combination with cisplatin. Methods. Cell lines used: SK-UT-1, SK-UT-1b and SK-LMS-1, the cytotoxicity of seliciclib and cisplatin was measured by the MTT assay. In combination with cisplatin the effects of seliciclib were examined by isobologram analysis. CDK2 levels were examined at mRNA and protein level by immunoblotting and PCR. We also looked at the effects of seliciclib on p53-dependent response of cells to seliciclib using immunoblotting. The effects of combination treatment were analysed using annexin V and PI staining by flow cytometric analysis. Results. IC 50 values for seliciclib were 10.5, 7.1 and 25.7 μM, for SK-UT-1, SK-UT-1b and SK-LMS-1 respectively, P53 in the SK-UT-1b (wild-type) and SK-LMS-1 lines (mutant) showed a wild-type response with induction seen with seliciclib treatment for 24 and 48 h. Seliciclib caused a decrease in CDK2 mRNA and protein over 72 h. A combination of cisplatin and seliciclib was synergistic in all three cell lines. Effects of combination treatment were an enhancement in apoptosis as judged by the emergence of a sub-G 1 population in cell cycle analysis and a sub-G 1 population with PI staining. Conclusions. Our data demonstrate the effectiveness of seliciclib as a single agent and when used in combination with cisplatin where the effects are synergistic.

Design and synthesis of novel amino-substituted xanthenones and benzo[ b]xanthenones: Evaluation of their antiproliferative activity and their ability to overcome multidrug resistance toward MES-SA/D × 5 cells

A number of new xanthenone and benzo[ b]xanthenone amino derivatives and their pyrazole-fused counterparts have been designed and synthesized possessing structural analogy to the potent anticancer agent 9-methoxypyrazoloacridine. The synthesis of the compounds proceeds through nucleophilic substitution of 1-chloro-4-nitroxanthenone or the corresponding benzo[ b]xanthenone by the appropriately substituted amine or hydrazine, reduction of the nitro group, and conversion into the suitable dialkylaminoacetamides. This method cannot be applied for synthesis of the pyrazole-fused benzo[ b]xanthenones, consequently a different, simple, and high-yielding synthetic procedure was developed for the preparation of the target molecules. In vitro cytotoxic potencies of the new derivatives toward the murine leukemia L1210 cell line, human colorectal adenocarcinoma (HT-29), and human uterine sarcoma (MES-SA and its 100-fold resistant to doxorubicin variant MES-SA/D × 5) cell lines are described and compared to those of reference drugs. The compounds exhibited significant cytotoxic activity against the tested cell lines and in addition they retain activity against the multidrug resistant MES-SA/D × 5 subline, showing resistant factors close to 1. A number of derivatives were found to posses DNA binding capacity, according to a standard ethidium bromide displacement assay. The majority of the studied compounds induce a G2/M arrest, although among them some G1 or S blockers have also been identified.

Characterisation of uterine sarcoma cell lines exhibiting MDR phenotype by vibrational spectroscopy

Multidrug resistance (MDR) enables cancer cells to escape cytotoxic insults of anticancer drugs. Rapid identification of cells exhibiting the MDR phenotype is very important since it can lead to an effective and individual patient based treatment plan. We have investigated a combined vibrational spectroscopic approach, using both micro-Raman and FTIR techniques, in order to characterise a sensitive human uterine sarcoma cell line MES-SA and its multidrug-resistant derivative Garf. In this study, these two complementary methods have been evaluated via the use of principal components analysis (PCA), for discrimination of cells exhibiting the MDR phenotype. Our results indicate that, though they inherently have different sensitivities, both Raman and IR methods can provide a good differentiation of cell phenotypes.

인체 자궁암 세포에서 천연 성분이 P-당단백질의 활성에 미치는 영향

Multidrug resistance (MDR) of cancer cells is, at least in part, associated with the overexpression of P-glycoprotein (P-gp). Many studies have demonstrated that natural compounds obtained from fruits, vegetables, teas and medicinal plants may modulate P-gp activity. The objective of the present investigation was to examine the effect of seven natural compounds on the P-gp activity in human uterine sarcoma cell line, MES-SA/DX5. Daunomycin uptake was significantly increased by biochanin A and silymarin (p value of daunomycin (p

Stable suppression of MDR-1 gene using siRNA expression vector to reverse drug resistance in a human uterine sarcoma cell line

Objective. Chemotherapy is highly effective in treating a number of gynecologic malignancies; however, its effectiveness diminishes with repeated exposures due to the emergence of multi-drug resistance (MDR). The aim of this study was to establish a permanent MDR gene knockdown model via infection with the siRNA-hairpin expression vector. The impact of transfecting the RNAi upon MDR-1 mRNA and P-glycoprotein expression as well as resultant chemotherapy resistance was assessed. Methods. Multi-drug resistant cell line MES-SA/DX5 was transfected with the siRNA-hairpin expression vector (pSMDR-HYG) designed to target MDR-1 mRNA. A negative control was established utilizing a vector lacking the anti-sense component (pSCON-HYG). The LD 50 of doxorubicin for the stable transfectants was determined utilizing a cytotoxic MTT assay. The mRNA expression of MDR-1 gene among those cell lines was evaluated by semi-quantitative RT-PCR. The product of P-glycoprotein (P-gp) was examined by Western blotting hybridization and immunostaining. Results. Two stable transfected cell lines: MES-SA/DX5-M (with pSMDR-HYG) and MES-SA/DX5-C (with pSCON-HYG) were established. The cell line MES-SA/DX5-M was nearly 7 times more sensitive to doxorubicin than MES-SA/DX5-C and its parent cell line MES-SA/DX5 ( P < 0.01). The mRNA expression of the MDR-1 gene in MES-SA/DX5-M was also statistically significantly lower than in the other 2 cell lines ( P < 0.01) as assessed by semi-quantitative RT-PCR. A barely detectable signal for P-gp (170 kDa) was observed in MES-SA/DX5-M. The vast majority of MES-SA/DX5-M cells were immunohistochemically negative for P-gp. Conclusions. Stable, sequence-specific MDR-1 gene silencing can be demonstrated by inducing the endogenous expression of hairpin siRNA. Hairpin-siRNA-based MDR-1 gene silencing correlated with decreased levels of MDR-1 mRNA and P-gp, thereby restoring permanent native chemosensitivity. This methodologic strategy may have significant clinical impact in reversing chemo-resistance, especially the multi-drug-resistant phenotype, in the treatment of gynecologic malignancies.

도노마이신의 세포 외 배출 및 세포 독성에 미치는 플라보노이드의 효과

One mechanism which plays a prominent role in development of multi-drug resistance seen in cancer cells is the over-expression of P-glycoprotein (P-gp). It is known that compounds found in vegetables and fruits not only have anticarcinogenic properties but may also modulate P-gp activity. The effect of some dietary components on efflux of daunomycin (DNM), a P-gp substrate, was examined in P-gp over-expressed human uterine sarcoma cell line, MES-SA/DX5. The efflux of DNM from the cells was significantly inhibited by quercetin and verapamil, but not by 1-naphtyl-isothiocyanate (NITC). The values for DNM in MES-SA/DX5 cells were increased by flavonoids (quercetin and fisetin), but not by NITC after 72 hour incubation with dietary constituents. In conclusion, flavonoids may play a role in the modulation of P­-gp activity in human uterine sarcoma cells.

인체 자궁암 세포에서 플라보노이드에 의한 P-당단백질의 활성 조절

One of the possible mechanisms of multi-drug resistance found in cancer cells is the over-expression of P­glycoprotein (P-gp). Studies have shown that compounds in plants including vegetables and fruits not only have anticancer activities but may also modulate P-gp activity. The effect of flavonoids and organic isothiocyanate on P-gp activity was studied in human uterine sarcoma cell lines, MES-SA (sensitive) and MES-SA/DX5 (resistant) cells. The accumulation of daunomycin (DNM), a P-gp substrate, was approximately 10 times greater in the sensitive cell as compared to the resistant cells over the entire time course (up to 2 hours). The positive control, verapamil increased the two hour accumulation of DNM while quercetin decreased that of DNM in the resistant cells. 1-Naphtyl-isothiocyanate (NITC) showed no effect on the two hour accumulation of DNM. The values for DNM in the resistant cells was about 20 times higher than that observed in the sensitive cells . Verapamil reduced the value for DNM whereas flavonoids (quercetin and fisetin) increased those for DNM in the resistant cells.

WTH3, a new member of the Rab6 gene family, and multidrug resistance

The WTH3 gene was obtained by a DNA fragment isolated by the methylation-sensitive representational difference analysis technique due to its hypermethylation in the human multidrug resistant (MDR) breast cancer cell line MCF7/AdrR. The WTH3 gene product is 89% and 91% identical to the human Rab6 and Rab6c proteins, but possesses an elongated C-terminal region which contains 46 extra amino acids. Nevertheless, we consider the WTH3 gene a new member of the Rab6 gene family. Semi-quantitative reverse transcriptase–polymerase chain reaction results showed that WTH3 was 15 and 4 times downregulated in MCF7/AdrR and MES-SA/Dx5, a human MDR uterine sarcoma cell line, as compared to their non-MDR parental cell lines. Permanent expression of the WTH3 transgene in MDR cell lines increased to varying degrees their sensitivity to several anticancer drugs, which included doxorubicin, taxol, vinblastine, vincristine, and etoposide, as compared to the control sublines transfected with the empty vector. Flow cytometry and fluorescence microscope experiments suggest that the WTH3 transgene stimulated the host’s uptake and retention of DOX. Our results imply that the WTH3 gene plays a role(s) in MDR phenotype development in vitro.

Rab6c, a new member of the Rab gene family, is involved in drug resistance in MCF7/AdrR cells

A new Rab6 homolog cDNA, Rab6c, was discovered by a hypermethylated DNA fragment probe that was isolated from a human multidrug resistant (MDR) breast cancer cell line, MCF7/AdrR, by the methylation sensitive-representational difference analysis (MS-RDA) technique. Rab6c was found to be under-expressed in MCF7/AdrR and MES-SA/Dx5 (a human MDR uterine sarcoma cell line) compared with their non-MDR parental cell lines. MCF7/AdrR cells expressing the exogenous Rab6c exhibited less resistance to several anti-cancer drugs, such as doxorubicin (DOX), taxol, vinblastine, and vincristine, than the control cells containing the empty vector. Flow cytometry experiments confirmed that the transfectants' diminished resistance to DOX was caused by increased drug accumulation induced by the exogenous Rab6c. These results indicate that Rab6c is involved in drug resistance in MCF7/AdrR cells.

Lysosomal accumulation of drugs in drug-sensitive MES-SA but not multidrug-resistant MES-SA/Dx5 uterine sarcoma cells.

Sequestration of drugs in intracellular vesicles has been associated with multidrug-resistance (MDR), but it is not clear why vesicular drug accumulation, which depends upon intracellular pH gradients, should be associated with MDR. Using a human uterine sarcoma cell line (MES-SA) and a doxorubicin (DOX)-resistant variant cell line (Dx-5), which expresses p-glycoprotein (PGP), we have addressed the relationship between multidrug resistance, vesicular acidification, and vesicular drug accumulation. Consistent with a pH-dependent mechanism of vesicular drug accumulation, studies of living cells vitally labeled with multiple probes indicate that DOX and daunorubicin (DNR) predominately accumulate in lysosomes, whose lumenal pH was measured at < 4.5, but are not detected in endosomes, whose pH was measured at 5.9. However, vesicular DOX accumulation is more pronounced in the drug-sensitive MES-SA cells and minimal in Dx5 cells even when cellular levels of DOX are increased by verapamil treatment. While lysosomal accumulation of DOX correlated well with pharmacologically induced differences in lysosome pH in MES-SA cells, lysosomal accumulation was minimal in Dx5 cells regardless of lysosomal pH. We found no differences in the pH of either endosomes or lysosomes between MES-SA and Dx5 cells, suggesting that, in contrast to other MDR cell systems, the drug-resistant Dx5 cells are refractory to pH-dependent vesicular drug accumulation. These studies demonstrate that altered endomembrane pH regulation is not a necessary consequence of cell transformation, and that vesicular sequestration of drugs is not a necessary characteristic of MDR.

Protein Kinase C Isoform Expression and Activity Alter Paclitaxel Resistancein Vitro

Objective.The aim of this study was to assess the relationship of protein kinase C (PKC) isoform expression and functional activity to the development of multidrug resistance in gynecologic malignancies.Methods.Paclitaxel-resistant subclones (T30 and T30-Res) of the Mes-sa human uterine sarcoma cell line were selected through exposure to paclitaxelin vitro.Indices of relative drug resistance were determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. Differences in the expression pattern of PKC isoforms were assessed by Western blot of cell lysates. Finally, the influence of PKC activity (i.e., translocation to the plasma membrane, confirmed by Western blot of plasma membrane bound protein) on resistance to paclitaxel was examined with the MTT assay in cells preincubated with PMA.Results.The indices of relative paclitaxel resistance of Mes-sa, Mes-sa-T30, and Mes-sa-T30-Res were 1-, 5-, and 11-fold, respectively. Five (α, γ, ι, λ, and μ) of the 11 known PKC isoforms were detected in all cell lysates. Only PKC-α and PKC-γ expression increased with increasing indices of paclitaxel resistance. Interestingly, PMA induction of PKC activity reversed resistance to paclitaxel in all cell lines by 2- to 3-fold, and this reversal of drug resistance was associated with a time-dependent translocation of PKC-α and PKC-γ to the plasma membrane compartment.Conclusions.Increased expression of only the PKC-α and PKC-γ isoforms correlates with increasing levels of paclitaxel resistance in Mes-sa cells in thisin vitroexperimental model. However, increased functional activity of these and other PKC isoforms leads to reversal in paclitaxel resistance. Therefore, PKC activating mechanisms normally present in primary tumor cells may be compromised in drug-resistant clones.

Doxorubicin selection for MDR1/P-glycoprotein reduces swelling-activated K+ and Cl- currents in MES-SA cells.

To test the hypothesis that P-glycoprotein enhances swelling currents through regulation of volume-sensitive Cl- channels [recently termed VSOAC (volume-sensitive osmolyte and anion channel)], a human uterine sarcoma cell line (MES-SA) and its doxorubicin-selected counterpart (Dx5) were studied. P-glycoprotein mRNA and protein levels were detected only in Dx5 cells. However, whole cell patch-clamp experiments showed that swollen Dx5 cells (n = 5) produced smaller VSOAC currents than MES-SA cells (n = 4; 106 +/- 26 pA/pF vs. 232 +/- 76 pA/pF at 90 mV). In radioisotopic efflux experiments, both swelling-activated 125I (Cl-) currents (n = 15) and 86Rb (K+) currents (n = 8) were found to be two-to fourfold smaller in the Dx5 (high P-glycoprotein) cells. Inhibitors of P-glycoprotein showed no specificity for the doxorubicin-selected cells (Dx5). Dideoxyforskolin (100 microM) blocked swelling-activated 125I efflux equally in both cell lines, whereas 100 microM verapamil had no effect. Thus, in this cell line, selection for P-glycoprotein expression is associated with reduced swelling currents. These findings suggest that P-glycoprotein expression does not directly facilitate VSOAC.