In the presence of BSO, As2O3 induced significant caspase-3 activation in all three cell lines in a time-dependent manner (Figure 6). At 48 h, the caspase-3 activity was increased 9.7-, 4.4- and 11.8-fold for NTUB1, NTUB1/P and NTUB1/As, respectively, as compared to the controls (allpo0.001). Among the three cell lines, caspase-3 levels were significantly higher in NTUB1/As than in the other two cells after Figure 3. Induction of the internucleosomal DNA frag-mentation in transitional carcinoma cells by As2O3with (A) or without (B) BSO. Concurrent treatment of As2O3
and BSO for 48 h induced evident apoptotic DNA frag-mentation in NTUB1/As at 24 h and in all three cell lines at 48 h, while treatment with As2O3alone brought on only minimal DNA fragmentation. M: marker (at 100 bp increments). C: control.
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activation at 24 and 48 h. These data indicate that As2O3-mediated apoptosis in the presence of BSO was associated with the activation of the caspase-3 cascade in transitional carcinoma cells.
Discussion
Although arsenic compounds have been applied clinically in the treatment of APL, little is known about its potential use in transitional carcinomas.
Cells exposed to arsenic compounds in vitro, typically As2O3, go through conventional apoptotic pathways that can be found in many tissue systems.
Our data showed that As2O3also induced apoptosis in transitional carcinoma cells.
Since most regimens of first-line systemic che-motherapy for advanced or metastatic transitional tumors are cisplatin-based, the possible cross-resis-tance between cisplatin and any second-line che-motherapeutic agents should be taken into consideration before the initiation of salvage treat-ment. Our data indicated that As2O3, especially in the
presence of BSO, showed significant activity against not only sensitive transitional carcinoma cells but also those resistant to cisplatin. Evident apoptotic events can be readily induced in these resistant cells by the combined treatment of As2O3plus BSO. This may warrant further investigations on its role in the salvage therapy for cisplatin-refractory transitional carcinomas.
Accumulating evidence showed that As2O3 -in-duced apoptosis involves classical pathways that are associated with ROS inductive signals.12 In brief, As2O3 elicits ROS production, rapid collapse in mitochondrial membrane potential, release of cyto-chrome c, caspase-3 activation, DNA fragmentation and, finally, morphologic evidence of apoptosis.
However, in prostate and ovarian cancer cell models, it was shown that As2O3-mediated cytotoxicity did not involve superoxide generation.7As2O3-mediated apoptotic pathways have never been explored in transitional carcinoma before. We have shown that the As2O3-induced apoptosis in transitional carcino-ma cells also went through the classical pathway as shown by the appearance of the sub-G1fraction and Figure 4. Flow cytometric analyses of relative amounts of ROSinduced in three transitional carcinoma cell lines by As2O3with or without BSO. Solid histograms (a) indicate the controls (treatment with drug-free medium for 18 h) and open histograms indicate treatment with As2O3for18 h.While As2O3alone brought on little ROSinduction, combined treatments with As2O3and BSO induced greater amounts of ROS in all three cell lines. Ma and Mb: the mean £uores-cence intensity of histograms a and b, respectively.
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internucleosomal DNA fragmentation upon exposure to As2O3. The upstream events included the produc-tion of ROS, loss of mitochondrial membrane
potential and activation of caspase-3. These events may take place in a sequence as shown here since arsenic-mediated ROS production occurred at as early as 18–24 h, yet the caspase-3 activity was not seen in NTUB1 and NTUB1/P until 48 h, and in NTUB1/As until 24 h after As2O3 exposure. The apoptotic events can be demonstrated not only in parental cells, but also in cells that are resistant to cisplatin or arsenic. Of note, cells treated with As2O3 and BSO showed a significantly greater extent of apoptosis than those with As2O3 alone within the same treatment duration. Since As2O3-mediated apoptosis is time dependent, all three cell lines would show evident apoptosis with longer exposure to As2O3alone. We only used one parental cell line in the present study. Additional studies using more transitional carcinoma cell lines are needed to generalize the notion that transitional carcinoma is sensitive to As2O3.
In conclusion, As2O3may serve as an active agent against human transitional carcinoma. As2O3 exerts its cytotoxic effect via the conventional apoptotic pathway that involves ROS production, loss of mitochondrial membrane potential, activation of Figure 5. Flow cytometric analyses of relative levels of the mitochondrial membrane potential in three transitional carcinoma cells treated with As2O3with or without BSO. Solid histograms (a) indicate the controls (treatment with drug-free medium for 48 h) while open histogramsindicate treatment with As2O3for 48 h.Reduction of the membrane potential was greater with the combined treatments than with As2O3alone. Ma and Mb: the mean £uorescence intensity of the histograms a and b, respectively.
Figure 6. Relative caspase-3 activity induced by As2O3
in the presence of BSO. At 48 h, the caspase-3 activity was increased 9.7-, 4.4- and 11.8-fold for NTUB1, NTUB1/
P and NTUB1/As, respectively, as compared to the con-trols (all po0.001). Data are presented as mean7SEM of three separate experiments.
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caspase-3 and internucleosomal DNA breakdown.
Our results have clinical implications and represent one of the few efforts to substantiate the clinical use of arsenic compounds in the treatment of human transitional carcinomas.
References
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2. Chen GQ, Zhu J, Shi XG, et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins.
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(Received 27 November 2001; accepted 10 December 2001)
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Resistance to Paclitaxel Is Proportional to Cellular Total Antioxidant Capacity
Balakrishnan Ramanathan,
1Kun-Yan Jan,
1Chien-Hung Chen,
1Tzyh-Chyuan Hour,
3Hong-Jen Yu,
2and Yeong-Shiau Pu
21Institute of Cellular and Organismic Biology, Academia Sinica;2Department of Urology, National Taiwan University College of Medicine, Taipei, Taiwan; and3Institute of Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan
Abstract
Paclitaxel, one of the most commonly prescribed chemo-therapeutic agents, is active against a wide spectrum of human cancer. The mechanism of its cytotoxicity, however, remains controversial. Our results indicate that paclitaxel treatment increases levels of superoxide, hydrogen peroxide, nitric oxide (NO), oxidative DNA adducts, G2-M arrest, and cells with fragmented nuclei. Antioxidants pyruvate and selenium, the NO synthase inhibitor NW-nitro-L-arginine methyl ester, and the NO scavenger manganese (III) 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide all decreased paclitaxel-mediated DNA damage and sub-G1
cells. In contrast, the glutamylcysteine synthase inhibitor buthionine sulfoximine (BSO) and the superoxide dismutase (SOD) inhibitor 2-methoxyestradiol (2-ME) increased the sub-G1 fraction in paclitaxel-treated cells. These results suggest that reactive oxygen and nitrogen species are involved in paclitaxel cytotoxicity. This notion is further supported with the observation that concentrations of paclitaxel required to inhibit cell growth by 50% correlate with total antioxidant capacity. Moreover, agents such as arsenic trioxide (As2O3), BSO, 2-ME, PD98059, U0126 [mitogen-activated protein/extracellular signal-regulated ki-nase inhibitors], and LY294002 (phosphatidylinositol 3-kinase/Akt inhibitor), all of which decrease clonogenic survival, also decrease the total antioxidant capacity of paclitaxel-treated cells, regardless whether they are pacli-taxel sensitive or paclipacli-taxel resistant. These results suggest that paclitaxel chemosensitivity may be predicted by taking total antioxidant capacity measurements from clinical tumor samples. This, in turn, may then improve treatment outcomes by selecting out potentially responsive patients.
(Cancer Res 2005; 65(18): 8455-60)
Introduction
Paclitaxel, originally isolated from Taxus brevifolia (pacific yew), is one of the most active chemotherapeutic agents against a wide panel of solid tumors including urothelial, breast, lung, and ovarian cancers (1, 2). The mechanism of paclitaxel cytotoxicity, however, remains controversial. Paclitaxel promotes the stable assembly of microtubules from a- and h-tubulin heterodimers and inhibits their de-polymerization (3). Thus, the antitumor effects of this drug
may result from interference with the normal function of micro-tubules and from blocking of cell cycle progression in late G2-M phases (4). Paclitaxel-induced apoptosis in hepatoma cells is mediated through G2-M arrest and DNA fragmentation (5). Cells with a defective G1checkpoint and with an increased percentage of G2-M fractions were found to have increased sensitivity to paclitaxel (6–8). However, the observation that in some cell lines, pulsed paclitaxel exposures causes apoptosis but not G2-M arrest suggests that paclitaxel-induced apoptosis may occur without a prior G2-M arrest (9). Moreover, paclitaxel has been shown to induce apoptosis in G1and S stages, but induce both apoptosis and necrosis in G2-M phase (10).
Paclitaxel has been reported to induce the formation of reactive oxygen species (ROS) and alter mitochondrial membrane perme-ability (11). Reduction of ROS by catalase or ascorbic acid treatment, however, does not correlate with the reduction of cytotoxicity in the human herpes virus 8–related tumor cell line BCBL-1, suggesting that oxidative stress is only partially involved in paclitaxel cytotoxicity (12). Moreover, treatment of the human T-cell lymphoblastic leukemia cell line CCRF-HSB-2 with the antioxidant N-acetyl-L-cysteine showed inhibition of paclitaxel-induced ROS production but did not prevent paclitaxel-paclitaxel-induced apoptosis, indicating that paclitaxel-induced apoptosis in these cells is ROS independent (13). In murine bladder tumor MBT-2 cells, paclitaxel has also been shown to activate a macrophage-mediated antitumor mechanism through a nitric oxide (NO)–
dependent pathway (14). Cotreating the human myeloid leukemia cell line HL-60 with paclitaxel and the NO-generating agent S-nitrosoglutathione decreases the accumulation of G2-M fractions, suggesting that NO prevents paclitaxel-treated cells from entering the G2-M phase (15).
The current study reveals our investigation into the role(s) of ROS and reactive nitrogen species in paclitaxel toxicity. Results support our hypothesis that ROS and reactive nitrogen species are involved in paclitaxel-induced apoptosis. We further show that in a wide panel of human cancer cell lines, cellular total antioxidant capacity is a critical determinant of cellular sensitivity to paclitaxel.
Materials and Methods
Cells.Cell lines MCF-7 and HCC1937 were cultured in DMEM. H460, H1299, H1355, SC-M1, HR, NTUB1 (16), and BFTC905 (17) were cultured in RPMI 1640. SV-HUC-1, 293, and T24 were cultured in F-12 medium; BEAS-2B was cultured in LHC-9 medium (BioSource International, Inc., Camarillo, CA); T24/A (18) was cultured in RPMI 1640 containing 0.4 Amol/L doxorubicin. NTUB1/P and NTUB1/T were maintained in RPMI 1640 containing 14 Amol/L cisplatin and 5 nmol/L paclitaxel, respectively (19).
All growth media were supplemented with 10% FCS, penicillin (100 units/
mL), streptomycin (100 Ag/mL), and 0.03% glutamine. Cultures were maintained at 37jC in a water-saturated atmosphere containing 5% CO2. Requests for reprints:Yeong-Shiau Pu, Department of Urology, National Taiwan
University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan. Phone: 886-2-23123456 ext. 5249; Fax: 886-2-23219145; E-mail: [email protected].
I2005 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-1162
www.aacrjournals.org 8455 Cancer Res 2005; 65: (18). September 15, 2005