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Side population is increased in paclitaxel-resistant ovarian cancer cell lines regardless of resistance to cisplatin
Gynecologic Oncology 121 (2011) 390–394
Contents lists available at ScienceDirect
Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
Side population is increased in paclitaxel-resistant ovarian cancer cell lines regardless of resistance to cisplatin Yoichi Kobayashi a,b,⁎, Ken-ichiro Seino c,g, Shinji Hosonuma a, Tatsuru Ohara a, Hiroaki Itamochi d, Seiji Isonishi e, Tsunekazu Kita f, Haruka Wada g, Satoshi Kojo c, Kazushige Kiguchi a a
Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Japan Department of Obstetrics and Gynecology, Kyorin University, School of Medicine, Japan Institute for Genetic Medicine Research Section of Pathophysiology, Hokkaido University, Japan d Department of Obstetrics and Gynecology, Tottori University, Japan e Department of Obstetrics and Gynecology, Jikei University, Japan f Department of Obstetrics and Gynecology, National Defense Medical College, Japan g Division of Bioregulation Research, Institute of Medical Science, St Marianna University, Japan b c
a r t i c l e
i n f o
Article history: Received 19 October 2010 Available online 26 January 2011 Keywords: Side population Paclitaxel-resistance Ovarian cancer Interferon-α
a b s t r a c t Objectives. In recent years, cancer stem cells (CSCs) have been reported to be correlated with chemoresistance and may also be enriched in side populations (SPs). In this study, the relationship between resistance to paclitaxel (PTX) and cisplatin (CDDP) and side populations was examined in three parental PTXand CDDP-sensitive ovarian cancer cell lines (2008, KF28, and TU-OM-1) and several other cell lines derived from these as well as the additional effects of interferon-alpha (INF-α). Methods. SP of three different parental cell lines and PTX- and/or CDDP-resistant cell lines derived from these was analyzed with flow cytometry. The expression of ABCB1 and ABCG2 in KF28 and its derived cell lines was examined. Additional cell-death effect of INF-α with PTX was also examined. Results. In the three parental cell lines and the PTX-sensitive cell lines derived from these lines, SP was very low. Conversely, in PTX-resistant cell lines, regardless of CDDP resistance, SP increased. ABCB1 was strongly expressed in the PTX-resistant cells, but not in their parental lines, which are sensitive to PTX. While INF-α showed only slight enhancement of the cell-death effect of PTX in PTX-sensitive cells, INF-α itself strongly induced apoptosis in PTX-resistant cells regardless of PTX concentration. Conclusions. The SP could be correlated with resistance to PTX. SP could be a target of INF-α, and resistance to PTX might be overcome by INF-α. © 2011 Elsevier Inc. All rights reserved.
Introduction In recent years, stem cell research has been developing rapidly in the fields of tissue regeneration and cancer stem cells (CSCs). CSCs are described as “multipotent cells capable of forming heterogeneous tumors in immunodeficient mice at high efficacy” [1]. CSCs were first identified in acute myeloid leukemia [2], and since then, CSCs have been found in many types of solid tumors including ovarian cancer [3–5]. Although some surface markers are used to isolate CSCs, no specific markers are known to prospectively identify CSCs in many types of solid tumors [6]. One of the characteristics to identify CSCs is the capability to exclude dyes such as Hoechst 33342 [6]. The Hoechst efflux phenomenon has proven to be a highly useful primary
⁎ Corresponding author. Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Fax: +81 422 47 3177. E-mail address: [email protected] (Y. Kobayashi). 0090-8258/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2010.12.366
purification strategy for isolating potential stem/progenitor cells from various tissues in the absence of cell-surface markers [7]. These cells are defined as a side population (SP). Common epithelial ovarian cancer is increasing in frequency, and its initial therapy is debulking surgery followed by platinum-taxan combined chemotherapy. Because more than half of the ovarian cancer cases are advanced, postoperative chemotherapy should play an important role. Although many patients may achieve complete remission, a majority of them may become resistant to the drugs and eventually relapse [8]. Therefore, overcoming chemoresistance is key to the treatment of ovarian cancer. The existence of a SP has been proven to be one of the reasons for chemoresistance because these cells express various types of ABC transporter genes [9,10]. Thus, it is very important to clarify the relationship between SPs and chemoresistance to overcome such drug resistance [10]. In this study, SPs were analyzed in three different parental ovarian cancer cell lines and the PTX- and/or CDDP-resistant cell lines derived from these cells, and the relationship between chemoresistance and SP is discussed.
Y. Kobayashi et al. / Gynecologic Oncology 121 (2011) 390–394
Materials and methods Cell lines Three different parental cell lines (2008, KF28, and TU-OM-1) and PTX- and/or CDDP-resistant cell lines (C13, 2008/PX24, KFr13, KF28TX, KFr13TX, TU-OM-1 CDDP, TU-OM-1 TX, and TU-OM-1 CDDP/TX) derived from these parental cell lines were used. The cell line 2008 was established from serous carcinoma of the ovary, and C13 and 2008/PX24 were derived from 2008 as cisplatin-resistant and
a
PTX-resistant cell lines, respectively [11]. The cell line KF28 was also established from serous carcinoma of the ovary, and KFr13, KF28TX, and KFr13TX were derived as cisplatin-resistant, paclitaxel-resistant, and both cisplatin- and paclitaxel-resistant cell lines, respectively [12]. The cell line TU-OM-1 was established from mucinous adenocarcinoma of the ovary, and TU-OM-1 CDDP, TU-OM-1 TX, and TU-OM-1 CDDP/TX were derived as cisplatin-resistant, TAXresistant, and both cisplatin- and TAX-resistant cell lines, respectively [13]. The cells were maintained in RPMI-1640 (Sigma, MO: for 2008 and KF series) and DMEM-F-12/HAM 1:1 vol/vol (Sigma-Aldrich, MO:
KF28 TX
KFr13
KFr13 TX
0.08
45.01
0.13
33.17
0.17
0.63
0.40
0.52
Hoechst Blue
Verapamil(+)
Verapamil(-)
KF28
391
Hoechst Red
b
2008
2008/PX24
C13
23.80
00.00
0.00
0.03
0.00
Hoechst Blue
Verapamil(+)
Verapamil(-)
0.00
Hoechst Red Fig. 1. Panels a, b, and c show the flow cytometric histograms of parental cell lines (KF 28, 2008, and TU-OM-1) and cell lines derived from their parental cells. Upper columns are histograms without verapamil, and lower columns are with verapamil. Numerical values in each histogram show the percentage of SP cells. Horizontal axis shows intensity of Hoechst blue, and vertical one shows that of Hoechst red. Side population is noted in paclitaxel-resistant cell lines in each figure.
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Fig. 1 (continued).
for TU-OM-1 series) with 10% FBS (Hyclone, Canada) and 1% Antibiotic–Antimycotic (Invitrogen, CA) in a humidified atmosphere containing 5% CO2 at 37 °C. Cell culture and detection of SPs After harvesting the cells with 0.5% trypsin-EDTA (Gibco, CA), 1 × 106 cells were conditioned in 1 ml of culture medium. Two milliliters of the cell-containing medium were incubated for 10 min at 37 °C, and then 10 μl of Hoechst 33342 (Sigma: final concentration 5 μl/ml) was added. One milliliter of cell-containing medium was transferred to another tube containing 50 μM of verapamil (Sigma) and was incubated for 80 min at 37 °C without exposure to light. Verapamil is known as one of the ATP-binding cassette blockers, and it blocks dye (Hoechs 33342) efflux. So, cells in side population fraction should markedly decrease by adding verapamil, and SP could be recognized by this procedure. The cells were then centrifuged at 1500 ×g for 5 min, and 300–400 μl of cold HBSS was added. Four microliters of propidium iodide (PI) (final concentration: 2 μl/ml) was added, and then the cells were analyzed with JSAN desktop cell sorter (Bay bioscience Co., Kobe, Japan). Determination of ABCB1 and ABCG2 gene expression by reverse transcription-polymerase chain reaction (RT-PCR) Expression of ABCB1 and ABCG2 was examined in KF28 and its derived cell lines (KFr13, KF28TX, and KFr13TX). Total cellular RNA was isolated using RNeasy (Qiagen Inc., CA). RNA was reversetranscribed using SuperScript III (Invitrogen, CA). A total of 100 ng of cDNA from each sample was amplified by polymerase chain reaction (PCR) using AmpliTaq Gold DNA Polymerase (Applied Biosystems). Detection of ABCB1 was performed with the following primers: forward primer, 5′-TGGGGCTTTTAGTGTTGGAC-3′; reverse primer, 5′CCTGACTCACCACACCAATG-3′. ABCG2 was detected with the following primers: forward primer, 5′-AGCTGCAAGGAAAGATCCAA-3′; reverse primer, 5′-CCTGCTTGGAAGGCTCTATG-3′. Amount of transcripts between each sample was normalized by glyceraldehyde-3phosphate dehydrogenase (G3PDH). The housekeeping gene G3PDH
was detected with the following forward primer, 5′-ACAGTCAGCCGCATCTTCTT-3′ and reverse primer, 5′-TTGATTTTGGAGGGATCTCG-3′. The PCR parameters were as follows: initial heating at 95 °C for 5 min.; followed by 30 cycles of denaturation at 95 °C for 30 s., annealing at 60 °C for 30 s., and extension at 72 °C for 60 s.; and a final extension at 72 °C for 5 min. The PCR products were electrophoresed in 1.5% agarose gels using 1× TAE buffer and stained with ethidium bromide (Sigma). The gels were photographed under UV light. Apoptotic effect of interferon-α in paclitaxel-resistant cells To examine the cytotoxic-effect of interferon (IFN)-α, KF28 (parental cells) and KF28TX (PTX-resistant cells derived from KF28) were incubated with 1000 U/ml of INF-α (Sumiferon 300: Dainippon pharmaceutical, Tokyo, Japan) for 7 days and then with various concentrations of PTX for 7 days. After that, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-annexin V (Beckman Coulter Inc.) and PI (Sigma) for fluorescence-activated cell sorting (FACS). These cells were analyzed using FC500 (Beckman Coulter Inc.) and FlowJo software (TreeStar Inc., OR), and the fraction of apoptotic cells was evaluated. Statistical analysis Each experiment was performed in triplicate, and non-parametric test was employed to obtain significance values. P-values less than 0.05 were considered statistically significant. Results SPs in various cell lines The results of representative experiments by flow cytometry are shown in Fig. 1. For example, the upper column of KF28TX in Fig. 1a shows 45.01% of cells are contained in the gate (side population; SP), which are markedly decreased after administration of 50 μM verapamil (Fig. 1a, KF28TX, lower column: 0.63%). SP could be determined with the ability of efflux of Hoechst 33342 due to its
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Discussion
Fig. 2. This figure shows expression of ABCB1 and ABCG2 in KF28 and cell lines derived from KF28. ABCB1 was strongly expressed in paclitaxel-resistant cell lines (KF28TX and KFr13TX).
expression of ABC transporter, and those cell populations are recognized as SP if they are diminished after administration of verapamil, a blocker of ABC transporter. Although SPs were scarcely detected in parental cell lines (2008, KF28, and TU-OM-1), SPs were enriched in PTX-resistant cell lines (2008/PX24, KF28TX, KFr13TX, and TU-OM-1 TX) compared to their parental cells. However, SPs were not enriched in CDDP-resistant cell lines (KFr13, C13, and TU-OM-1 CDDP). In both PTX- and CDDPresistant cell lines (KFr13TX, TU-OM-1 CDDP/TX), SP enrichment also increased significantly. Thus, regardless of CDDP resistance, SP enrichment increased only in PTX-resistant cell lines.
Expression of ABCB1/ABCG2 and correlation with chemoresistance As shown in Fig. 2, ABCB1 was strongly expressed in KF28TX and KFr13TX, which acquire paclitaxel-resistance, but not in the parental KF28 and cisplatin-resistant KFr13 cells, which are sensitive to PTX. ABCG2 was expressed in all cell lines tested but was strongly expressed in KF28TX.
Apoptotic effect of interferon (IFN)-α in paclitaxel-resistant cells Fig. 3 shows the effect of PTX with or without INF-α in KF28 and KF28TX cells. In KF28 cells, PTX induced significant cell-death at low concentrations (0.0005 nM, p b 0.05) compared to cells without exposure to INF-α, but its effect was minimal. In KF28TX cells, INF-α itself strongly induced apoptosis in PTX-resistant cells, but additional dose-dependent effects of PTX were not observed in all concentrations tested in the cells exposed to INF-α.
CSCs have been reported to be one of the reasons for recurrence or relapse of various cancers because CSCs are resistant to anti-cancer drugs or radiation therapy [14]. Although cytotoxic reagents may seem to be effective and could lead to complete remission, CSCs could survive and result in relapse or metastasis, ultimately leading to the patients' death. Overcoming the CSC population could be one of the keys to conquering this lethal disease. Several groups have reported the existence of SPs in ovarian cancer. Szotek et al. detected SPs in human ovarian cancer cell lines and in primary ascites cancer cells [4], and Hirschmann-Jax et al. reported that these SPs expressed high levels of the transporter genes ABCG2 and ABCA3 and showed that SPs are resistant to cytotoxic drugs [15]. In ovarian cancer, SPs could be detected and may contribute to drug resistance. Moreover, SP cells in ovarian cancer were reported to have CSC characteristics, showing that CSCs could be enriched in SPs in ovarian cancer [4,16]. Hu L et al. reported that the proportion of SPs in various human ovarian cancer cell lines was relatively low, ranging from 0.1 to 2.4% [16]. Here, we showed that the percentage of SPs was also low in three different parental cell lines (2008, KF28, and TU-OM1). However, our study also revealed that SPs were significantly enriched in the parental-derived PTX-resistant cell lines. In our present study, SPs were enriched in three different paclitaxel-resistant cell lines compared to their parental cell lines. Conversely, the fraction of SP cells was the same in cisplatin-resistant paclitaxel-sensitive cell lines compared to their parental cell lines. We also showed that ABCB1 was strongly expressed in paclitaxel-resistant cell lines, although it was not expressed in paclitaxel-sensitive parental cells or cisplatin-resistant cells. Because PTX resistance has been reported to correlate with expression of ABC transporters such as ABCB1 and ABCC2 [17], it seems reasonable that the size of the SP is increased in PTX-resistant cells. Indeed, the expression of ABCB1 was increased in PTX-resistant cell lines (KF28TX and KFr13TX) and was not detected in PTX-sensitive cell lines. PTX-resistance may therefore be correlated with SP, but CDDP-resistance likely is not. Thus, targeting of SPs could overcome PTX-resistance but not CDDPresistance, and the mechanism of resistance to CDDP should be correlated to something other than SP enrichment. Expression of ABCG2 was weaker in a PTX- and CDDP-resistant cell line (KFr13TX) than a PTX-resistant CDDP-sensitive cell line (KF28TX). Yamamoto et al. reported sensitivity to CDDP increased in a CDDP-resistant ovarian cancer cell line (KFr13) after inducing PTX-resistance [18]. Resistance to PTX and CDDP may therefore influence each other.
Fig. 3. This figure shows additional effect of paclitaxel in KF28 and KF28TX incubated with interferon-α. Horizontal axis shows concentration of PTX, and vertical axis shows percentage of survival cells.IFN-α: interferon-α, PTX: paclitaxel.
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INF-α has been used to treat patients with various types of cancer [19–22]. Moserle et al. reported that SPs could be a target of INF-α in ovarian cancer cell lines [23]. Interestingly, they also showed INF-α treatment caused a dramatic reduction in SP size in cell lines bearing a large SP fraction in vitro. Here, we also revealed that INF-α itself apparently induces apoptosis in PTX-resistant KF28TX cells, an ovarian cancer cell line with a large SP fraction. Moserle et al. also reported that several genes, such as IFI16, could be involved in the anti-proliferative and pro-apoptotic activities exerted by INF-α on SP cells; however, they found that INF-α did not modify the expression of ABCG2 and MDR1 transporters, which led them to speculate that other unknown mechanisms are involved in modulating the IFN signal [23]. This study revealed that the SP was increased in PTX-resistant ovarian cancer cell lines but was not increased in PTX-sensitive cell lines regardless of CDDP-resistance. To overcome chemoresistance in ovarian cancer, SP might represent a viable target in PTX-resistant patients. The correlation between CDDP-resistance and CSCs needs to be further examined. Though there are many challenges to overcome resistance to PTX and CDDP, success could ultimately lead to a drastically improved prognosis of patients with epithelial ovarian cancer. Conflict of interest statement There is no conflict of interest for all of the authors.
Acknowledgment
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[12]
[13]
[14] [15]
[16] [17] [18]
The authors are grateful to Ms. Kaori Aida and Mr. Hajime Kamijuku for their skillful technical supports.
[19]
References [20] [1] Polyak K, Hahn WC. Roots and stems: stem cells in cancer. Nat Med 2006;12: 296–300. [2] Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994;367:645–8. [3] Bapat SA, Mali AM, Koppikar CB, Kurrey NK. Stem and progenitor-like cells contribute to the aggressive behavior of human ovarian cancer. Cancer Res 2005;65:3025–9. [4] Szotek P, Pieretti-Vanmarche R, Masiakos P, Dinulescu D, Connolly D, Foster R, et al. Ovarian cancer side population defines cells with stem cell-like character-
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istics and Mullerian-inhibiting substance responsiveness. Proc Natl Acad Sci USA 2006;103:11154–9. Zhang S, Balch C, Chan MW, Lai H, Matei D, Schilder JM, et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 2008;68:4311–20. Wu C, Alman BA. Side population cells in human cancers. Cancer Lett 2008;268: 1–9. Challen GA, Little MH. A side order of stem cells: the SP phenotype. Stem Cells 2006;24:3–12. Pan Y, Huang X. Epithelial ovarian cancer stem cells — a review. Int J Clin Exp Med 2008;1:260–6. Fong MY, Kakar SS. The role of cancer stem cells and the side population in epithelial ovarian cancer. Histol Histopathol 2010;25:113–20. Hadnagy A, Gaboury L, Beaulieu R, Balicki D. SP analysis may be used to identify cancer stem cell populations. Exp Cell Res 2006;312:3701–10. Isonishi S, Saitou M, Yasuda M, Ochiai K, Tanaka T. Enhancement of sensitivity to cisplatin by orobol is associated with increased mitochondrial cytochrome c release in human ovarian carcinoma cells. Gynecol Oncol 2003;90:413–20. Ishibashi M, Nakayama K, Yeasmin S, Katahiri A, Iida K, Nakayama N, et al. A BTB/ POZ gene, NAC-1, a tumor recurrence-associated gene, as a potential target for Taxol resistance in ovarian cancer. Clin Cancer Res 2008;14:3149–55. Sato S, Itamochi H, Kigawa J, Oishi T, Shimada M, Sato S, et al. Combination chemotherapy of oxaliplatin and 5-fluorouracil may be an effective regimen for mucinous adenocarcinoma of the ovary: a potential treatment strategy. Cancer Sci 2009;100:546–51. Hemmings C. The elaboration of a critical framework for understanding cancer: the cancer stem cell hypothesis. Pathology 2010;42:105–12. Hirshmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U, et al. A distinct “side population” of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA 2004;101:14228–33. Hu L, McArthur C, Jaffe RB. Ovarian cancer stem-like side-population cells are tumorigenic and chemoresistant. Br J Cancer 2010;102:1276–83. Stravrovskaya AA, Stromskaya TP. Transport proteins of the ABC family and multidrug resistance of tumor cells. Biochemistry (Moscow) 2008;73:592–604. Yamamoto K, Kikuchi Y, Kudoh K, Nagata I. Modulation of cisplatin sensitivity by taxol in cisplatin-sensitive and -resistant human ovarian carcinoma cell lines. J Cancer Res Clin Oncol 2000;126:168–72. Morschella F, Proiette E, Capone I, Belardelli F. Combination strategies for enhancing the efficacy of immunotherapy in cancer patients. Ann NY Acad Sci 2010;1194:169–78. Mocellin S, Pasquali S, Rossi CR, Nitti D. Interferone alpha adjuvant therapy in patients with high-risk melanoma: a systematic review and meta-analysis. J Natl Cancer Inst 2010;102:493–501. Tirone NR, Michelin MA, Murta EF. Using cytokine to treat cervical intraepithelial and invasive neoplasia. Recent Pat Anticancer Drug Discov 2010;1:165–9. Tagliaferri P, Caraglia M, Budillon A, Marra M, Vitale G, Viscomi C, et al. New pharmacokinetic and pharmacodynamic tools for interferon- (IFN-) treatment of human cancer. Cancer Immunol Immunother 2005;54:1–10. Moserle L, Indraccolo S, Ghisi M, Frasson C, Fortunato E, Canecari S, et al. The side population of ovarian cancer cells is a primary target of INF-α antitumor effects. Cancer Res 2008;68:5658–68.
Contents lists available at ScienceDirect
Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
Side population is increased in paclitaxel-resistant ovarian cancer cell lines regardless of resistance to cisplatin Yoichi Kobayashi a,b,⁎, Ken-ichiro Seino c,g, Shinji Hosonuma a, Tatsuru Ohara a, Hiroaki Itamochi d, Seiji Isonishi e, Tsunekazu Kita f, Haruka Wada g, Satoshi Kojo c, Kazushige Kiguchi a a
Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Japan Department of Obstetrics and Gynecology, Kyorin University, School of Medicine, Japan Institute for Genetic Medicine Research Section of Pathophysiology, Hokkaido University, Japan d Department of Obstetrics and Gynecology, Tottori University, Japan e Department of Obstetrics and Gynecology, Jikei University, Japan f Department of Obstetrics and Gynecology, National Defense Medical College, Japan g Division of Bioregulation Research, Institute of Medical Science, St Marianna University, Japan b c
a r t i c l e
i n f o
Article history: Received 19 October 2010 Available online 26 January 2011 Keywords: Side population Paclitaxel-resistance Ovarian cancer Interferon-α
a b s t r a c t Objectives. In recent years, cancer stem cells (CSCs) have been reported to be correlated with chemoresistance and may also be enriched in side populations (SPs). In this study, the relationship between resistance to paclitaxel (PTX) and cisplatin (CDDP) and side populations was examined in three parental PTXand CDDP-sensitive ovarian cancer cell lines (2008, KF28, and TU-OM-1) and several other cell lines derived from these as well as the additional effects of interferon-alpha (INF-α). Methods. SP of three different parental cell lines and PTX- and/or CDDP-resistant cell lines derived from these was analyzed with flow cytometry. The expression of ABCB1 and ABCG2 in KF28 and its derived cell lines was examined. Additional cell-death effect of INF-α with PTX was also examined. Results. In the three parental cell lines and the PTX-sensitive cell lines derived from these lines, SP was very low. Conversely, in PTX-resistant cell lines, regardless of CDDP resistance, SP increased. ABCB1 was strongly expressed in the PTX-resistant cells, but not in their parental lines, which are sensitive to PTX. While INF-α showed only slight enhancement of the cell-death effect of PTX in PTX-sensitive cells, INF-α itself strongly induced apoptosis in PTX-resistant cells regardless of PTX concentration. Conclusions. The SP could be correlated with resistance to PTX. SP could be a target of INF-α, and resistance to PTX might be overcome by INF-α. © 2011 Elsevier Inc. All rights reserved.
Introduction In recent years, stem cell research has been developing rapidly in the fields of tissue regeneration and cancer stem cells (CSCs). CSCs are described as “multipotent cells capable of forming heterogeneous tumors in immunodeficient mice at high efficacy” [1]. CSCs were first identified in acute myeloid leukemia [2], and since then, CSCs have been found in many types of solid tumors including ovarian cancer [3–5]. Although some surface markers are used to isolate CSCs, no specific markers are known to prospectively identify CSCs in many types of solid tumors [6]. One of the characteristics to identify CSCs is the capability to exclude dyes such as Hoechst 33342 [6]. The Hoechst efflux phenomenon has proven to be a highly useful primary
⁎ Corresponding author. Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Fax: +81 422 47 3177. E-mail address: [email protected] (Y. Kobayashi). 0090-8258/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2010.12.366
purification strategy for isolating potential stem/progenitor cells from various tissues in the absence of cell-surface markers [7]. These cells are defined as a side population (SP). Common epithelial ovarian cancer is increasing in frequency, and its initial therapy is debulking surgery followed by platinum-taxan combined chemotherapy. Because more than half of the ovarian cancer cases are advanced, postoperative chemotherapy should play an important role. Although many patients may achieve complete remission, a majority of them may become resistant to the drugs and eventually relapse [8]. Therefore, overcoming chemoresistance is key to the treatment of ovarian cancer. The existence of a SP has been proven to be one of the reasons for chemoresistance because these cells express various types of ABC transporter genes [9,10]. Thus, it is very important to clarify the relationship between SPs and chemoresistance to overcome such drug resistance [10]. In this study, SPs were analyzed in three different parental ovarian cancer cell lines and the PTX- and/or CDDP-resistant cell lines derived from these cells, and the relationship between chemoresistance and SP is discussed.
Y. Kobayashi et al. / Gynecologic Oncology 121 (2011) 390–394
Materials and methods Cell lines Three different parental cell lines (2008, KF28, and TU-OM-1) and PTX- and/or CDDP-resistant cell lines (C13, 2008/PX24, KFr13, KF28TX, KFr13TX, TU-OM-1 CDDP, TU-OM-1 TX, and TU-OM-1 CDDP/TX) derived from these parental cell lines were used. The cell line 2008 was established from serous carcinoma of the ovary, and C13 and 2008/PX24 were derived from 2008 as cisplatin-resistant and
a
PTX-resistant cell lines, respectively [11]. The cell line KF28 was also established from serous carcinoma of the ovary, and KFr13, KF28TX, and KFr13TX were derived as cisplatin-resistant, paclitaxel-resistant, and both cisplatin- and paclitaxel-resistant cell lines, respectively [12]. The cell line TU-OM-1 was established from mucinous adenocarcinoma of the ovary, and TU-OM-1 CDDP, TU-OM-1 TX, and TU-OM-1 CDDP/TX were derived as cisplatin-resistant, TAXresistant, and both cisplatin- and TAX-resistant cell lines, respectively [13]. The cells were maintained in RPMI-1640 (Sigma, MO: for 2008 and KF series) and DMEM-F-12/HAM 1:1 vol/vol (Sigma-Aldrich, MO:
KF28 TX
KFr13
KFr13 TX
0.08
45.01
0.13
33.17
0.17
0.63
0.40
0.52
Hoechst Blue
Verapamil(+)
Verapamil(-)
KF28
391
Hoechst Red
b
2008
2008/PX24
C13
23.80
00.00
0.00
0.03
0.00
Hoechst Blue
Verapamil(+)
Verapamil(-)
0.00
Hoechst Red Fig. 1. Panels a, b, and c show the flow cytometric histograms of parental cell lines (KF 28, 2008, and TU-OM-1) and cell lines derived from their parental cells. Upper columns are histograms without verapamil, and lower columns are with verapamil. Numerical values in each histogram show the percentage of SP cells. Horizontal axis shows intensity of Hoechst blue, and vertical one shows that of Hoechst red. Side population is noted in paclitaxel-resistant cell lines in each figure.
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Fig. 1 (continued).
for TU-OM-1 series) with 10% FBS (Hyclone, Canada) and 1% Antibiotic–Antimycotic (Invitrogen, CA) in a humidified atmosphere containing 5% CO2 at 37 °C. Cell culture and detection of SPs After harvesting the cells with 0.5% trypsin-EDTA (Gibco, CA), 1 × 106 cells were conditioned in 1 ml of culture medium. Two milliliters of the cell-containing medium were incubated for 10 min at 37 °C, and then 10 μl of Hoechst 33342 (Sigma: final concentration 5 μl/ml) was added. One milliliter of cell-containing medium was transferred to another tube containing 50 μM of verapamil (Sigma) and was incubated for 80 min at 37 °C without exposure to light. Verapamil is known as one of the ATP-binding cassette blockers, and it blocks dye (Hoechs 33342) efflux. So, cells in side population fraction should markedly decrease by adding verapamil, and SP could be recognized by this procedure. The cells were then centrifuged at 1500 ×g for 5 min, and 300–400 μl of cold HBSS was added. Four microliters of propidium iodide (PI) (final concentration: 2 μl/ml) was added, and then the cells were analyzed with JSAN desktop cell sorter (Bay bioscience Co., Kobe, Japan). Determination of ABCB1 and ABCG2 gene expression by reverse transcription-polymerase chain reaction (RT-PCR) Expression of ABCB1 and ABCG2 was examined in KF28 and its derived cell lines (KFr13, KF28TX, and KFr13TX). Total cellular RNA was isolated using RNeasy (Qiagen Inc., CA). RNA was reversetranscribed using SuperScript III (Invitrogen, CA). A total of 100 ng of cDNA from each sample was amplified by polymerase chain reaction (PCR) using AmpliTaq Gold DNA Polymerase (Applied Biosystems). Detection of ABCB1 was performed with the following primers: forward primer, 5′-TGGGGCTTTTAGTGTTGGAC-3′; reverse primer, 5′CCTGACTCACCACACCAATG-3′. ABCG2 was detected with the following primers: forward primer, 5′-AGCTGCAAGGAAAGATCCAA-3′; reverse primer, 5′-CCTGCTTGGAAGGCTCTATG-3′. Amount of transcripts between each sample was normalized by glyceraldehyde-3phosphate dehydrogenase (G3PDH). The housekeeping gene G3PDH
was detected with the following forward primer, 5′-ACAGTCAGCCGCATCTTCTT-3′ and reverse primer, 5′-TTGATTTTGGAGGGATCTCG-3′. The PCR parameters were as follows: initial heating at 95 °C for 5 min.; followed by 30 cycles of denaturation at 95 °C for 30 s., annealing at 60 °C for 30 s., and extension at 72 °C for 60 s.; and a final extension at 72 °C for 5 min. The PCR products were electrophoresed in 1.5% agarose gels using 1× TAE buffer and stained with ethidium bromide (Sigma). The gels were photographed under UV light. Apoptotic effect of interferon-α in paclitaxel-resistant cells To examine the cytotoxic-effect of interferon (IFN)-α, KF28 (parental cells) and KF28TX (PTX-resistant cells derived from KF28) were incubated with 1000 U/ml of INF-α (Sumiferon 300: Dainippon pharmaceutical, Tokyo, Japan) for 7 days and then with various concentrations of PTX for 7 days. After that, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-annexin V (Beckman Coulter Inc.) and PI (Sigma) for fluorescence-activated cell sorting (FACS). These cells were analyzed using FC500 (Beckman Coulter Inc.) and FlowJo software (TreeStar Inc., OR), and the fraction of apoptotic cells was evaluated. Statistical analysis Each experiment was performed in triplicate, and non-parametric test was employed to obtain significance values. P-values less than 0.05 were considered statistically significant. Results SPs in various cell lines The results of representative experiments by flow cytometry are shown in Fig. 1. For example, the upper column of KF28TX in Fig. 1a shows 45.01% of cells are contained in the gate (side population; SP), which are markedly decreased after administration of 50 μM verapamil (Fig. 1a, KF28TX, lower column: 0.63%). SP could be determined with the ability of efflux of Hoechst 33342 due to its
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Discussion
Fig. 2. This figure shows expression of ABCB1 and ABCG2 in KF28 and cell lines derived from KF28. ABCB1 was strongly expressed in paclitaxel-resistant cell lines (KF28TX and KFr13TX).
expression of ABC transporter, and those cell populations are recognized as SP if they are diminished after administration of verapamil, a blocker of ABC transporter. Although SPs were scarcely detected in parental cell lines (2008, KF28, and TU-OM-1), SPs were enriched in PTX-resistant cell lines (2008/PX24, KF28TX, KFr13TX, and TU-OM-1 TX) compared to their parental cells. However, SPs were not enriched in CDDP-resistant cell lines (KFr13, C13, and TU-OM-1 CDDP). In both PTX- and CDDPresistant cell lines (KFr13TX, TU-OM-1 CDDP/TX), SP enrichment also increased significantly. Thus, regardless of CDDP resistance, SP enrichment increased only in PTX-resistant cell lines.
Expression of ABCB1/ABCG2 and correlation with chemoresistance As shown in Fig. 2, ABCB1 was strongly expressed in KF28TX and KFr13TX, which acquire paclitaxel-resistance, but not in the parental KF28 and cisplatin-resistant KFr13 cells, which are sensitive to PTX. ABCG2 was expressed in all cell lines tested but was strongly expressed in KF28TX.
Apoptotic effect of interferon (IFN)-α in paclitaxel-resistant cells Fig. 3 shows the effect of PTX with or without INF-α in KF28 and KF28TX cells. In KF28 cells, PTX induced significant cell-death at low concentrations (0.0005 nM, p b 0.05) compared to cells without exposure to INF-α, but its effect was minimal. In KF28TX cells, INF-α itself strongly induced apoptosis in PTX-resistant cells, but additional dose-dependent effects of PTX were not observed in all concentrations tested in the cells exposed to INF-α.
CSCs have been reported to be one of the reasons for recurrence or relapse of various cancers because CSCs are resistant to anti-cancer drugs or radiation therapy [14]. Although cytotoxic reagents may seem to be effective and could lead to complete remission, CSCs could survive and result in relapse or metastasis, ultimately leading to the patients' death. Overcoming the CSC population could be one of the keys to conquering this lethal disease. Several groups have reported the existence of SPs in ovarian cancer. Szotek et al. detected SPs in human ovarian cancer cell lines and in primary ascites cancer cells [4], and Hirschmann-Jax et al. reported that these SPs expressed high levels of the transporter genes ABCG2 and ABCA3 and showed that SPs are resistant to cytotoxic drugs [15]. In ovarian cancer, SPs could be detected and may contribute to drug resistance. Moreover, SP cells in ovarian cancer were reported to have CSC characteristics, showing that CSCs could be enriched in SPs in ovarian cancer [4,16]. Hu L et al. reported that the proportion of SPs in various human ovarian cancer cell lines was relatively low, ranging from 0.1 to 2.4% [16]. Here, we showed that the percentage of SPs was also low in three different parental cell lines (2008, KF28, and TU-OM1). However, our study also revealed that SPs were significantly enriched in the parental-derived PTX-resistant cell lines. In our present study, SPs were enriched in three different paclitaxel-resistant cell lines compared to their parental cell lines. Conversely, the fraction of SP cells was the same in cisplatin-resistant paclitaxel-sensitive cell lines compared to their parental cell lines. We also showed that ABCB1 was strongly expressed in paclitaxel-resistant cell lines, although it was not expressed in paclitaxel-sensitive parental cells or cisplatin-resistant cells. Because PTX resistance has been reported to correlate with expression of ABC transporters such as ABCB1 and ABCC2 [17], it seems reasonable that the size of the SP is increased in PTX-resistant cells. Indeed, the expression of ABCB1 was increased in PTX-resistant cell lines (KF28TX and KFr13TX) and was not detected in PTX-sensitive cell lines. PTX-resistance may therefore be correlated with SP, but CDDP-resistance likely is not. Thus, targeting of SPs could overcome PTX-resistance but not CDDPresistance, and the mechanism of resistance to CDDP should be correlated to something other than SP enrichment. Expression of ABCG2 was weaker in a PTX- and CDDP-resistant cell line (KFr13TX) than a PTX-resistant CDDP-sensitive cell line (KF28TX). Yamamoto et al. reported sensitivity to CDDP increased in a CDDP-resistant ovarian cancer cell line (KFr13) after inducing PTX-resistance [18]. Resistance to PTX and CDDP may therefore influence each other.
Fig. 3. This figure shows additional effect of paclitaxel in KF28 and KF28TX incubated with interferon-α. Horizontal axis shows concentration of PTX, and vertical axis shows percentage of survival cells.IFN-α: interferon-α, PTX: paclitaxel.
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INF-α has been used to treat patients with various types of cancer [19–22]. Moserle et al. reported that SPs could be a target of INF-α in ovarian cancer cell lines [23]. Interestingly, they also showed INF-α treatment caused a dramatic reduction in SP size in cell lines bearing a large SP fraction in vitro. Here, we also revealed that INF-α itself apparently induces apoptosis in PTX-resistant KF28TX cells, an ovarian cancer cell line with a large SP fraction. Moserle et al. also reported that several genes, such as IFI16, could be involved in the anti-proliferative and pro-apoptotic activities exerted by INF-α on SP cells; however, they found that INF-α did not modify the expression of ABCG2 and MDR1 transporters, which led them to speculate that other unknown mechanisms are involved in modulating the IFN signal [23]. This study revealed that the SP was increased in PTX-resistant ovarian cancer cell lines but was not increased in PTX-sensitive cell lines regardless of CDDP-resistance. To overcome chemoresistance in ovarian cancer, SP might represent a viable target in PTX-resistant patients. The correlation between CDDP-resistance and CSCs needs to be further examined. Though there are many challenges to overcome resistance to PTX and CDDP, success could ultimately lead to a drastically improved prognosis of patients with epithelial ovarian cancer. Conflict of interest statement There is no conflict of interest for all of the authors.
Acknowledgment
[5]
[6] [7] [8] [9] [10] [11]
[12]
[13]
[14] [15]
[16] [17] [18]
The authors are grateful to Ms. Kaori Aida and Mr. Hajime Kamijuku for their skillful technical supports.
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