In ovarian cancer patients, the duration of complete response after successful cancer chemotherapy is often short-lived. During that time period, patients usually receive no further maintenance therapy. Ovarian cancer patients in complete remission, however, often begin to show steady increases in their plasma levels of cancer antigen CA-125, 4-6 months before the recurrence of clinical symptoms. For these individuals, we are proposing the administration of angiogenesis inhibitors as a maintenance therapy. Although, in ovarian cancer, CA-125 provides a unique opportunity in identifying those patients requiring further treatment, this concept could be applied to other forms of cancer as other prognostic indicators become available.
Survivin is a novel anti-apoptotic protein that is highly expressed in cancer but is undetectable in most normal adult tissues. It was reported that taxol-mediated mitotic arrest of cancer cells is associated with survivin induction, which preserves a survival pathway and results in resistance to taxol. In this study, we provide new evidence that induction of survivin by taxol is an early event and is independent of taxol-mediated G2/M arrest. Taxol treatment of MCF-7 cells rapidly up-regulated survivin expression (3.5–15-fold) within 4 h without G2/M arrest. Lengthening the treatment of cells (48 h) with taxol resulted in decreased survivin expression in comparison with early times following taxol treatment, although G2/M cells were significantly increased at later times. Interestingly, 3 nm taxol induces survivin as effectively as 300 nm and more effectively than 3000 nm. As a result, 3 nm taxol is ineffective at inducing cell death. However, inhibition of taxol-mediated survivin induction by small interfering RNA significantly increased taxol-mediated cell death. Taxol rapidly activated the phosphatidylinositol 3-kinase/Akt and MAPK pathways. Inhibition of these pathways diminished survivin induction and sensitized cells to taxol-mediated cell death. A cis-acting DNA element upstream of -1430 in the survivin pLuc-2840 construct is at least partially responsible for taxol-mediated survivin induction. Together, these data show, for the first time, that taxol-mediated induction of survivin is an early event and independent of taxol-mediated G2/M arrest. This appears to be a new mechanism for cancer cells to evade taxol-induced apoptosis. Targeting this survival pathway may result in novel approaches for cancer therapeutics. Survivin is a novel anti-apoptotic protein that is highly expressed in cancer but is undetectable in most normal adult tissues. It was reported that taxol-mediated mitotic arrest of cancer cells is associated with survivin induction, which preserves a survival pathway and results in resistance to taxol. In this study, we provide new evidence that induction of survivin by taxol is an early event and is independent of taxol-mediated G2/M arrest. Taxol treatment of MCF-7 cells rapidly up-regulated survivin expression (3.5–15-fold) within 4 h without G2/M arrest. Lengthening the treatment of cells (48 h) with taxol resulted in decreased survivin expression in comparison with early times following taxol treatment, although G2/M cells were significantly increased at later times. Interestingly, 3 nm taxol induces survivin as effectively as 300 nm and more effectively than 3000 nm. As a result, 3 nm taxol is ineffective at inducing cell death. However, inhibition of taxol-mediated survivin induction by small interfering RNA significantly increased taxol-mediated cell death. Taxol rapidly activated the phosphatidylinositol 3-kinase/Akt and MAPK pathways. Inhibition of these pathways diminished survivin induction and sensitized cells to taxol-mediated cell death. A cis-acting DNA element upstream of -1430 in the survivin pLuc-2840 construct is at least partially responsible for taxol-mediated survivin induction. Together, these data show, for the first time, that taxol-mediated induction of survivin is an early event and independent of taxol-mediated G2/M arrest. This appears to be a new mechanism for cancer cells to evade taxol-induced apoptosis. Targeting this survival pathway may result in novel approaches for cancer therapeutics. Survivin is a recently characterized novel member of the inhibitor of apoptosis (IAP) 1The abbreviations used are: IAP, inhibitor of apoptosis; siRNA, small interfering RNA; PI3K, phosphatidylinositol 3-kinase; Erk or ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TBS-T, Tris-buffered saline plus Tween 20; DMEM, Dulbecco's modified Eagle's medium. protein family. It is undetectable in most normal adult tissues but highly expressed in cancer. Survivin expression has been shown to be associated with carcinogenesis, cancer progression, poor prognosis, drug resistance, and short patient survival (1Li F. J. Cell. Physiol. 2003; 197: 8-29Crossref PubMed Scopus (307) Google Scholar) and that inhibition of survivin expression and/or function in tumor cells by survivin antisense or dominant-negative mutants triggers apoptosis (2Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1737) Google Scholar, 3Ambrosini G. Adida C. Sirugo G. Altieri D.C. J. Biol. Chem. 1998; 273: 11177-11182Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 4Grossman D. McNiff J.M. Li F. Altieri D.C. Lab. Investig. 1999; 79: 1121-1126PubMed Google Scholar, 5O'Connor D.S. Grossman D. Plescia J. Li F. Zhang H. Villa A. Tognin S. Marchisio P.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13103-13107Crossref PubMed Scopus (588) Google Scholar, 6Olie R.A. Simoes-Wust A.P. Baumann B. Leech S.H. Fabbro D. Stahel R.A. Zangemeister-Wittke U. Cancer Res. 2000; 60: 2805-2809PubMed Google Scholar) as well as a defect in cell division (7Li F. Ackermann E.J. Bennett C.F. Rothermel A.L. Plescia J. Tognin S. Villa A. Marchisio P.C. Altieri D.C. Nat. Cell Biol. 1999; 1: 461-466Crossref PubMed Scopus (554) Google Scholar, 8Chen J. Wu W. Tahir S.K. Kroeger P.E. Rosenberg S.H. Cowsert L.M. Bennett F. Krajewski S. Krajewska M. Welsh K. Reed J.C. Ng S.C. Neoplasia. 2000; 2: 235-241Crossref PubMed Scopus (184) Google Scholar). Thus, survivin is considered an exciting target for cancer prevention and therapeutics. Taxol (paclitaxel) is one of the most active cancer chemotherapeutic agents. It is effective against a variety of human tumors including ovarian, breast, and non-small-cell lung tumors as well as head and neck carcinomas (9McGuire W.P. Rowinsky E.K. Rosenshein N.B. Grumbine F.C. Ettinger D.S. Armstrong D.K. Donehower R.C. Ann. Intern. Med. 1989; 111: 273-279Crossref PubMed Scopus (1124) Google Scholar, 10Liebmann J.E. Cook J.A. Lipschultz C. Teague D. Fisher J. Mitchell J.B. Br. J. Cancer. 1993; 68: 1104-1109Crossref PubMed Scopus (388) Google Scholar, 11Rowinsky E.K. Donehower R.C. N. Engl. J. Med. 1995; 332: 1004-1014Crossref PubMed Scopus (1979) Google Scholar, 12Eisenhauer E.A. Vermorken J.B. Drugs. 1998; 55: 5-30Crossref PubMed Scopus (262) Google Scholar, 13Wiseman L.R. Spencer C.M. Drugs Aging. 1998; 12: 305-334Crossref PubMed Scopus (78) Google Scholar). However, its effectiveness is often limited because many tumors display taxol resistance. Cancer cells can acquire resistance to taxol by at least two different mechanisms (14Casazza A.M. Fairchild C.R. Cancer Treat. Res. 1996; 87: 149-171Crossref PubMed Scopus (43) Google Scholar). Overexpression of the multidrug resistance 1(MDR1) gene, which encodes P-glycoprotein, can confer resistance to taxol. This is because P-glycoprotein functions as a xenobiotic pump that pumps taxol as well as many other chemotherapeutic agents out of cells (15Bhalla K. Huang Y. Tang C. Self S. Ray S. Mahoney M.E. Ponnathpur V. Tourkina E. Ibrado A.M. Bullock G. Willingham M.C. Leukemia. 1994; 8: 465-475PubMed Google Scholar). The other is that tubulin mutations, which result in alterations in either the assembly or stability of microtubules, can lead to taxol resistance (16Schibler M.J. Cabral F. J. Cell Biol. 1986; 102: 1522-1531Crossref PubMed Scopus (110) Google Scholar, 17Giannakakou P. Sackett D.L. Kang Y.K. Zhan Z. Buters J.T. Fojo T. Poruchynsky M.S. J. Biol. Chem. 1997; 272: 17118-17125Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). However, taxol resistance resulting from apoptotic blockade has not been well studied. Taxol treatment induces mitotic arrest through taxol-induced polymerization and stabilization of microtubules (18Kumar N. J. Biol. Chem. 1981; 256: 10435-10441Abstract Full Text PDF PubMed Google Scholar, 19Schiff P.B. Fant J. Horwitz S.B. Nature. 1979; 277: 665-667Crossref PubMed Scopus (3149) Google Scholar, 20Schiff P.B. Horwitz S.B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1561-1565Crossref PubMed Scopus (1758) Google Scholar, 21Parness J. Horwitz S.B. J. Cell Biol. 1981; 91: 479-487Crossref PubMed Scopus (551) Google Scholar, 22Manfredi J.J. Parness J. Horwitz S.B. J. Cell Biol. 1982; 94: 688-696Crossref PubMed Scopus (359) Google Scholar), and it induces cell death by apoptosis or necrosis dependent of drug concentration (23Woods C.M. Zhu J. McQueney P.A. Bollag D. Lazarides E. Mol. Med. 1995; 1: 506-526Crossref PubMed Google Scholar, 24Ireland C.M. Pittman S.M. Biochem. Pharmacol. 1995; 49: 1491-1499Crossref PubMed Scopus (46) Google Scholar, 25Fan W. Biochem. Pharmacol. 1999; 57: 1215-1221Crossref PubMed Scopus (148) Google Scholar, 26Blagosklonny M.V. Robey R. Sheikh M.S. Fojo T. Cancer Biol. Ther. 2002; 1: 113-117Crossref PubMed Scopus (40) Google Scholar). On the other hand, it has been demonstrated that survivin expression is cell cycle-regulated with a robust increase in the G2/M phase of cell cycle (2Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1737) Google Scholar, 27Li F. Altieri D.C. Cancer Res. 1999; 59: 3143-3151PubMed Google Scholar). Presumably, cells treated with taxol should show an increased survivin expression because of G2/M arrest. Consistent with this notion, it was reported that taxol-induced microtubule stabilization and mitotic arrest increase survivin expression, which engenders a cell survival pathway to counteract taxol-induced apoptosis (28O'Connor D.S. Wall N.R. Porter A.C. Altieri D.C. Cancer Cell. 2002; 2: 43-54Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). However, it is not clear whether this mitotic survival pathway is the only means involving survivin by which cancer cells counteract taxol-induced apoptosis following drug treatment. Here, we report that induction of survivin by taxol is an early event and is independent of taxol-mediated G2/M arrest. We found that taxol treatment of MCF-7 breast cancer cells rapidly up-regulated survivin expression without apparent arrest of cells into G2/M phase. Lengthening the treatment of cells (48 h) with taxol resulted in decreased survivin expression in comparison with early times following taxol treatment, although the percentage of cells in G2/M phase was significantly increased at later times. Consistent with the observation that 3 nm taxol induced survivin as effectively as 300 nm and more effectively than 3000 nm taxol, 3 nm taxol are ineffective for apoptotic induction in these cells. However, inhibition of taxol-mediated induction of survivin by small interfering/inhibitory RNA (siRNA) significantly increased taxol-mediated cell death. Mechanistic studies indicated that taxol rapidly activated the PI3K/Akt and Erk MAPK pathways, and inhibition of PI3K/Akt signaling by Ly294002 or Erk MAPK signaling by U0126/PD98059 diminished survivin induction by taxol and sensitized cells to taxol-induced cell death. Survivin promoter-luciferase reporter assays revealed that early taxol-mediated induction of survivin is at least in part transcriptionally regulated and that the cis-acting DNA element mediating the effects of taxol on survivin promoter activity is located upstream of -1430 in the pLuc-2840 construct. Together, these data show for the first time that induction of survivin by taxol is an early event following drug exposure and is independent of taxol-mediated G2/M arrest. This appears to be a new mechanism by which cancer cells evade taxol-induced apoptosis. Targeting this novel survival pathway may have potential in cancer therapeutic applications. Cell Culture—MCF-7 cells, a breast cancer cell line without P-glycoprotein and multidrug-resistant protein-1 expression, were maintained in RPMI 1640 medium containing 10% fetal bovine serum (MediaTech CellGro, Herndon, VA), penicillin (100 units/ml), and streptomycin (0.1 μg/ml) (Invitrogen) in a humidified atmosphere incubator with 5% CO2 at 37 °C. Cells were routinely subcultured twice weekly. Reagents—Taxol, anti-β-actin, goat peroxidase-conjugated anti-rabbit IgG and fluorescein isothiocyanate-conjugated anti-rabbit IgG were purchased from Sigma. Anti-survivin (FL-142), anti-Erk (K-23), and anti-Akt (H-136) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-Akt (Ser-473) and phospho-p44/42 MAPK (Thr-202/Tyr-204) antibodies were purchased from Cell Signaling Technology (Beverly, MA). LY294002, PD98059, U0126, and dual luciferase report assay system were purchased from Promega (Madison, WI). Oligotransfectamine reagent was purchased from Invitrogen. FuGENE 6 transfection reagent was bought from Roche Diagnostics (Indianapolis, IN). Treatment—Taxol stock solutions were 4 mm in Me2SO and stored at -20 °C. Just prior to taxol treatment, the stock solution was freshly diluted with Me2SO (3, 30, 300, and 3000 μm) and then further diluted in RPMI 1640 medium to final concentrations of 3, 30, 300, and 3000 nm, respectively. In the concentration-dependent experiments, MCF-7 cells were treated with 3, 30, 300, and 3000 nm taxol for 8 h. In the time-dependent experiments, MCF-7 cells were treated with 30 nm taxol and harvested at 4, 8, 16, 24, and 48 h after taxol treatment. In the subcellular localization experiments, MCF-7 cells were treated with 3 nm taxol. For all of the experiments, the control group was treated with the same amount Me2SO. In experiments for exploration of signaling pathways and cell death, MCF-7 cells were treated with and without taxol (3 and 30 nm) in the presence and absence of various concentrations of PI3K (LY294002) or MEK (PD98059 and U0126) inhibitors. Survivin expression was analyzed by Western blot described below after treatment for 8 h. Cell morphological changes were microscopically photographed after treatment for 48 h. Western Blot—Cells with and without treatment were washed with phosphate-buffered saline (PBS: 50 mm phosphate, pH 7.4, 100 mm NaCl, and 10 mm KCl) and lysed on ice for 30 min in PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 μg/ml phenylmethylsulfonyl fluoride, and 20 μm leupeptin. After the lysates were cleared by centrifugation at 15,000 × g for 20 min at 4 °C, the total protein was determined using Bio-Rad protein assay solution. Up to 75 μg of total protein was denatured in 2× SDS sample loading buffer for 5 min at 95 °C, separated on 10–15% SDS-PAGE gels, and electrotransferred to Immobilon-P membranes (Millipore, Bedford, MA) using semidry electrophoretic transfer. After the nonspecific binding sites on the membranes were blocked with 5% skimmed milk or bovine serum albumin (BSA) in TBS-T (20 mm Tris-HCl, pH 7.5, 0.137 m NaCl, and 0.01% Tween 20) for 3 h at room temperature with constant shaking, the membranes were incubated in TBS-T containing the relevant primary antibody (1:500–1000) and 5% BSA overnight at 4 °C. After washing with TBS-T for three times, the membrane was incubated in 5% skimmed milk in TBS-T buffer containing the appropriate second anti-IgG antibody (1:5000) at room temperature for 1 h with constant shaking. The phosphorylation or expression of the target protein was detected by the ECL protein detection kit (Amersham Biosciences) following the manufacturer's instructions and visualized by autoradiography with the Kodak X-Omat AR film. For normalization of protein loading, the same membranes were stripped with stripping buffer (100 mm 2-mercaptoethanol, 2% SDS, and 62.5 mm Tris-HCl, pH 6.7) and used for Western blot by the same procedure with a monoclonal antibody against β-actin (1:1000 dilution) and/or with the polyclonal antibodies against the relevant total protein. Trypan Blue Exclusion Staining for Determination of Cell Viability— Cells to be counted were collected by trypsinization/centrifugation and resuspended in PBS buffer. A small sample of the cell suspension was diluted in 0.4% (w/v) trypan blue (one sample at a time because viable cells absorb trypan blue over time as well). A cover glass was centered over the hemacytometer chambers, and one chamber was filled with the cell dilution using a Pasteur pipette. Stained (dead) and unstained (viable) cells were counted in each of the four corner and central squares under an inverted microscope using ×100 magnification, respectively. Each cell sample was counted in this way for three times. The percentage of cell viabilities in each sample was calculated with the formula of % viability = total viable cell numbers/total cell numbers × 100. Propidium Iodide Staining and Flow Cytometry—At various time intervals after taxol treatment (30 nm) as described above, the cells were harvested by trypsinization and washed with PBS. Cells (∼1 × 106) were resuspended in 5 ml of 70% ethanol. After the initial fixation, cells were suspended in 0.5 ml of PBS containing 25 μg/ml propidium iodide, 0.2% Triton X-100, and 40 μg/ml RNase A and incubated for at least 30 min at 4 °C. The cells then were analyzed for immunofluorescence intensities by flow cytometry (FACScan, BD Biosciences) from 10,000 events/sample. Data from flow cytometry were analyzed using WinList software (Verity Software House Inc., Topsham, ME). For each time point, triplicate assays were performed. Immunofluorescence Microscopy—Cells were seeded on the round glass coverslips coated with 2% gelatin (Sigma) in 12-well plates. At different intervals after 3 nm taxol treatment as described above, medium was removed and cells were washed once with PBS and fixed overnight with 4% paraformaldehyde in PBS at 4 °C. Cells were then permeabilized and blocked for 30 min in PBS containing 2% BSA and 0.2% Triton X-100. For survivin and nuclear DNA double staining, the blocked cells were first incubated in PBS containing 1% BSA and rabbit anti-survivin antibody (1:500) for 60 min at 37 °C followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200) for 30 min at 37 °C. The nuclear DNA was then stained with 4′,6-diamidino-2-phenylindole at a final concentration of 0.5 μg/ml in H2O for 10 min at room temperature. The resultant glass coverslips containing cells were mounted on glass slides with Gel/Mount™ solution (Biomedia Corp., Foster City, CA). The cells were analyzed under a Zeiss Axiovert 100M digital fluorescence microscope. Images were captured using Zeiss LSM510, version 2.8 and processed with Photoshop Element software. SiRNA Preparation—A human survivin mRNA-specific RNA oligonucleotides with 3′-TT overhangs were chemically synthesized and purified by high pressure liquid chromatography (Xeragon, Huntsville, AL) as follows: SRi-2F (92GCG CCU GCA CCC CGG AGC G110TT*) and SRi-2R (110CGC UCC GGG GUG CAG GCG C92TT). Equal moles of SRi-2F/SRi-2R (designated SRi-2) were mixed together to a final concentration of 20 μm in annealing buffer (100 mm KAc, 30 mm HEPES-KOH, and 2 mm MgAc2, pH 7.4). After denaturation at 90 °C for 1 min, the mixture (Sri-2) was annealed at 37 °C for 60 min and stored at -80 °C for transfection experiments. A scramble RNA duplex (designated scraSRi) was also prepared same as above for a negative control in this study. The scramble sequence (5′-CAG UCG CGU UUG CGA CUG GTT-3′ (forward chain) and 5′-CCA GUC GCA AAC GCG ACU GTT-3′ (reverse chain)) was not present in mammalian cells by BLAST search at NCBI. In Vitro Transfection with siRNAs—Cells were transfected with survivin siRNAs using the Oligofectamine reagent following the manufacturer's instruction. 1 day prior to transfection, 5 × 104 MCF-7 cells/well were seeded in six-well plates (corresponding to a density of 40% at the time of transfection) without antibiotics. The transfection mixture was prepared by mixing 175 μl of DMEM containing 6 μl of 20 μm siRNA with 15 μl of DMEM containing 3 μl of Oligofectamine reagents. Before transfection, the medium in 6-well plates was replaced with serum-free DMEM medium (800 μl/well). The transfection mixture was added to the 6-well plate within 20–40 min after mixture preparation in a total volume of 990 μl/well. The transfected cells were incubated at 37 °C for 4 h, and then 500 μl of DMEM medium containing 30% fetal bovine serum was added. Cells were treated with and without 3 and 30 nm taxol 24 h after transfection with siRNA or control siRNA as described above. For Western blot, cells were harvested 8 h after taxol treatment. For flow cytometry analysis of sub-G1 DNA content, cells were harvested 24 and 48 h after taxol treatment and analyzed by propidium iodide staining and flow cytometry as described above. The percentage of dead cells (sub-G1 DNA contents) was plotted as a histogram. All of the transfection experiments were performed at triplicate for each experiment. Luciferase Reporter Assay—MCF-7 cells were transfected with survivin promoter-luciferase constructs pLuc-6270, which contains a 6.3-kb survivin promoter sequence (29Li F. Altieri D.C. Biochem. J. 1999; 344: 305-311Crossref PubMed Scopus (289) Google Scholar), using the FuGENE 6 transfection reagent (Roche Diagnostics), and luciferase activities were measured using a dual luciferase reporter assay system (Promega) according to the respective manufacturers' recommendations. Cells were seeded in 24-well plates 1 day prior to transfection. On the following day, the transfection solution for each well was prepared by sequentially adding 1 μl of FuGENE 6 transfection reagent and 0.4 μg of plasmid DNA in a 1.5-ml tube containing 50 μl of serum-free DMEM medium. The DNA FuGENE 6 mixture was incubated at room temperature for 20–30 min and then added onto cells at ∼50% confluence in each well containing complete medium. The transfected cells were treated with 3 and/or 30 nm taxol 24 h after transfection for the 30-h time point, and then taxol (3 and/or 30 nm in final concentration) was added to the transfected cells at other time points at 4 (26-h time point), 8 (22-h time point), and 22 h (8-h time point) after the first taxol treatment to permit the simultaneous harvesting of the cells from all of the time points. Cells were washed with PBS and lysed in 200 μl of 1× passive lysis buffer (Promega) per well 8 h after the last taxol treatment. After incubation of the plate on ice for 45 min, the lysate was transferred to a 1.5-ml Eppendorf tube by scraping with a rubber policeman. Cellular debris was pelleted by centrifugation at 15,000 × g for 10 min at 4 °C. 20 μl of cell lysates/well was used to measure Firefly and Renilla luciferase activity using a luminometer by adding 20 μl of luciferase assay reagent. Luciferase activity was normalized to Renilla luciferase activities as arbitrary units and plotted as a histogram from a representative experiment in triplicate. Induction of Survivin by Taxol Is an Early Event and Is Independent of taxol-mediated G2/M Arrest—It was previously shown that taxol (2–200 nm) treatment of HeLa cells for 48 h increases survivin expression to 1.2–4-fold in taxol-mediated G2/M arrest (28O'Connor D.S. Wall N.R. Porter A.C. Altieri D.C. Cancer Cell. 2002; 2: 43-54Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). To closely investigate the relationship between taxol-mediated G2/M arrest and survivin induction, MCF-7 cells were treated with taxol at various concentrations and times as shown (Fig. 1A) and survivin expression was analyzed by Western blot. To our surprise, lengthening taxol treatments (48 h or more) significantly attenuated survivin induction in comparison with the early times (24 h, Fig. 1A). The high induction of survivin at 24 h suggested that induction of survivin expression might occur at even earlier times. Consistent with this notion, Western blot experiments to test early time points after taxol treatment indicated that taxol-mediated survivin induction is as early as 4 h and that extending treatment times beyond 24 h diminished the effect of taxol on survivin induction in comparison with earlier time points (Fig. 1B). This result strongly suggested that induction of survivin by taxol is probably independent of taxol-mediated G2/M arrest. To confirm this possibility, we determined cell cycle distribution by propidium iodide staining and flow cytometry after taxol treatment. As shown (Fig. 2A), there is no apparent G2/M cell population increase at the 4 and 8-h times of taxol treatment in comparison with the no taxol control. A significant increase of G2/M cell population was observed only after taxol treatment for 16 h or more (Fig. 2A). We next examined individual cells by survivin immunofluorescence microscopy after taxol treatment for 4 h. The results indicated that interphase cells showed increased immunoreactivity with anti-survivin antibody in comparison to no taxol treatment control. A representative experiment from the immunofluorescence microscopy study is shown in Fig. 2B. The G2/M phase-independent induction of survivin expression by taxol was further confirmed by monitoring the expression of cyclin B1 (a G2/M phase marker) after taxol treatment. There was no change in cyclin B1 expression 4 h after taxol treatment (Fig. 2C). Interestingly, the cyclin B1 protein was almost undetectable 48 h after taxol treatment (Fig. 2C), suggesting that the cells were synchronized in later anaphase.Fig. 2Effects of taxol on cell cycle distribution and the expression of survivin and cyclin B1 in MCF-7 cells. Cells were analyzed by flow cytometry and immunofluorescence as described under "Experimental Procedures." A, cell cycle distribution was determined by propidium iodide (PI) staining and flow cytometry after taxol treatment at various time points and shown as a histogram. Each bar represents the mean ± S.D. from a representative experiment in triplicate. Cell viability determined by trypan blue exclusion in each time point is shown in Fig. 1B. B, rapid induction of survivin expression by taxol was determined by immunofluorescence microscopy. As shown, survivin expression in non-mitotic cells was significantly increased 4 h after taxol treatment in comparison with no taxol treatment control. C, time course of induction of cyclin B1 expression after taxol treatment. The relative-fold increase of cyclin B1 protein after normalization to β-actin internal control is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Low Concentrations (3–30 nm) of Taxol Effectively Induced Survivin Expression but Were Ineffective for Apoptosis Induction—Unexpectedly, the induction of survivin expression by taxol at low concentrations (3 nm) was as effective as or more effective than high concentrations (3000 nm) (Fig. 3A). Consistent with the observation that high expression of survivin increases cell viability and engenders drug resistance (28O'Connor D.S. Wall N.R. Porter A.C. Altieri D.C. Cancer Cell. 2002; 2: 43-54Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 30Zaffaroni N. Pennati M. Colella G. Perego P. Supino R. Gatti L. Pilotti S. Zunino F. Daidone M.G. Cell Mol. Life Sci. 2002; 59: 1406-1412Crossref PubMed Scopus (248) Google Scholar, 31Wall N.R. O'Connor D.S. Plescia J. Pommier Y. Altieri D.C. Cancer Res. 2003; 63: 230-235PubMed Google Scholar), low concentrations (3–30 nm) of taxol were ineffective for induction of cell death in comparison with high concentrations of the drug (Fig. 3B). Inhibition of Taxol-mediated Induction of Survivin Expression by siRNA-sensitized Taxol-induced Cell Death—We employed a survivin siRNA approach (32Ling X. Li F. BioTechniques. 2004; (in press)PubMed Google Scholar) for inhibition of taxol-mediated survivin induction to determine the effect of survivin inhibition on taxol-induced cell death. Fig. 4A shows the structures of siRNAs used in this study. Consistent with the result that survivin siRNA significantly inhibited the taxol-mediated induction of survivin in MCF-7 cells (Fig. 4B), propidium iodide staining and flow cytometry analysis indicated that a combination of siRNA-targeting survivin and a low concentration of taxol treatment strikingly increased cell death (the sub-G1 DNA content increase) in comparison with either treatment alone (Fig. 4C). This observation suggests that survivin expression plays a critical role in cell viability and that induction of survivin by taxol is a potential drug resistance factor leading to cell survival. Taxol Treatment Rapidly Enhanced Akt and Erk1/2 Phosphorylation in MCF-7 Cells—It has been shown that activation of the Akt survival pathway can up-regulate survivin expression (33Papapetropoulos A. Fulton D. Mahboubi K. Kalb R.G. O'Connor D.S. Li F. Altieri D.C. Sessa W.C. J. Biol. Chem. 2000; 275: 9102-9105Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar, 34Tran J. Master Z. Yu J.L. Rak J. Dumont D.J. Kerbel R.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4349-4354Crossref PubMed Scopus (415) Google Scholar) and inhibition of Akt and Erk1/2 activation can block growth factor-mediated induction of survivin (35Fukuda S. Foster R.G. Porter S.B. Pelus L.M. Blood. 2002; 100: 2463-2471Crossref PubMed Scopus (126) Google Scholar). To delineate the underlying mechanism by which taxol rapidly induced survivin expression, the activation of Akt and Erk1/2 after taxol treatment was examined in MCF-7 cells. Interestingly, the phosphorylation of both Akt and Erk1/2 were rapidly and strongly increased after taxol treatment of these cells (Fig. 5). Inhibition of Taxol-mediated PI3K/Akt Signaling by LY294002 or MEK/Erk Signaling by U0126/PD98059 Diminished Taxol-mediated S
Journal Article Book Reviews Get access Altered Glycosylation in Tumor Cells Christopher L. Reading, Sen-itiroh Hakomori, Donald M. Marcus eds. New York: Alan R. Liss, Inc., 1988, 332 pp. $140.00 RALPH J. BERNACKI RALPH J. BERNACKI Department of Experimental Therapeutics Grace Cancer Drug Center Roswell Park Memorial InstituteBuffalo, NY 14263 Search for other works by this author on: Oxford Academic PubMed Google Scholar JNCI: Journal of the National Cancer Institute, Volume 81, Issue 3, 1 February 1989, Page 232, https://doi.org/10.1093/jnci/81.3.232 Published: 01 February 1989
Clinical drug resistance is frequently associated with overexpression of the multidrug resistance (MDR) proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP). Taxanes are substrates for Pgp and MRP-1, but not BCRP. Taxane-based reversal agents (tRAs) are non-cytotoxic MDR modulators previously examined for broad-spectrum modulation of Pgp, MRP-1 and BCRP.Modulation by tRAs was studied by flow cytometry and resistance to taxanes was studied in cytotoxicity assays in the parental HL60/wt, 8226/wt and MCF7/S, and the resistant HL60/ADR, 8226/Dox6, 8226/MR20 and MCF7 AdVp3000 cell lines. Amino acid sequence (BLAST) alignments were performed using ClustalW.Structure-activity analysis demonstrated greatest alignment of BCRP with the transmembrane 7-12 region of Pgp and identified tRA side groups that contributed or were detrimental to modulation.Identification of tRA side groups contributing to modulation of Pgp, MRP-1 and BCRP will allow the design of a next generation of tRAs and will optimize their potential clinical applicability.
A glycoprotein : sialyltransferase which catalyzed the addition of N ‐acetyl[ 14 C]neuraminic acid to desialyzed glycoprotein precursors has been partially purified and characterized from rat liver, using desialyzed human prothrombin as an exogenous acceptor. The enzyme was extracted with 0.1% Triton X‐100 and was purified 31‐fold by gel chromatography. The sialyltransferase with desialyzed human prothrombin as acceptor was most active at 37 ° C, pH 7.0, 15 mM Mg 2+ , and its activity toward other asialoglycoproteins was similar with respect to optimum conditions. The enzyme was inhibited by 10 mM HgCl 2 , PbCl 2 and ZnCl 2 . Kinetic data were treated according to the model of Cleland for a bisubstrate reaction and the various constants were K b = 50 μM, K b = 31 μM, K ia = 10 μM, K ib = 5.8 μM, and V 1 = 100 nmol × h −1 × mg protein −1 , where a = CMP‐ N ‐acetyl[ 14 C]neuraminic acid and b = prothrombin. The activities with various desialyzed acceptors could not be separated by gel chromatography and may be due to a single sialyltransferase. Rats made hypothrombinemic by maintenance on a vitamin‐K‐deficient diet had normal or slightly elevated amounts of sialyltransferase activity using either desialyzed prothrombin or fetuin as glycoprotein acceptors for N ‐acetylneuraminic acid. Rat liver obtained from vitamin‐K‐deficient rats also had normal amounts of other glycosyltransferases, including galactosyl, glucosyl and N ‐acetylglucosaminyltransferase, as measured with endogenous or exogenous acceptors.
Proc Amer Assoc Cancer Res, Volume 46, 2005
1219
Characterization of Chiral Tamoxifen Analogs Tamoxifen (TAM), a non-steroidal selective estrogen receptor modulator (SERM), is used for the treatment of estrogen receptor (ER) positive breast cancer. There are limitations however to its usefulness including a 4-fold increase of endometrial carcinoma associated with long term usage in postmenopausal women and the development of resistance. Based on these problems we have synthesized and evaluate a series of new chiral tamoxifen analogs. These stereo-selective chiral derivatives differ from TAM by replacement of the central double bond with a cyclopropyl moiety, introducing two chiral centers within the molecule. We hypothesize that novel chiral TAM analogs will demonstrate tissue specific activity through ER isotype (α and β) selectivity as well as inducing unique ligand-ER complex conformations. Our studies aim to direct the syntheses of future chiral TAM analogs by determining what effects altering the size, hydrophobicity and stereochemistry of a molecule have on its interaction with ER isotypes. Once characterized, these analogs are not only potential therapeutic agents, but become tools with which to assess SERM-ER interactions. Growth inhibitory properties were analyzed using MCF7-S (ER+ breast cancer cell line), its sublines TAM resistant MCF7/LCC2, Faslodex resistant MCF7/LCC9 (Brunner et al., Cancer Res. 57:3486, 1997) and the ER- breast cancer cell line BT-20. Cell growth was assessed using the sulforhodamine B colorimetric assay. Analog 99149 was found to be 3-fold more potent than TAM and 99148 (its enantiomer) using MCF7-S, while 02009 was found to be 2-fold more potent than TAM against resistant LCC2 cells. Isolated ER competitive binding data correlates ERα binding affinity with growth inhibitive activity in MCF7-S cells. Similar experiments performed with ERβ did not correlate with cell growth inhibitory activity. This suggests that ERα plays a vital role in SERM growth inhibitory activity in MCF7-S cells. Reporter plasmid XETL containing an estrogen response element (ERE) linked to a luciferase gene, enabled the evaluation of direct inhibition of ERE mediated gene expression. MCF7-S cells were transiently transfected with XETL and treated with 100nM of E2 and/or 2μM of TAM or analogs. Agent 99149 was found to be less potent than TAM in inhibiting ERE mediated transcription. This does not directly correlate with the growth inhibitory effects suggesting that the greater antiproliferative activity seen with 99149 may be due to drug effects other than inhibition of ERE gene expression. Structure-function relationships are being analyzed within a set of ten tamoxifen analogs. (This work is supported by NCI Student Training Grant CA09072-28)
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
