Background
Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor, and the second leading cause of cancer-related deaths in the world [
1]. Over the past decade, advances in treatment, medical device development, surgical techniques, radiology, and liver transplantation have resulted in considerable improvements in therapy for HCC [
2,
3]. However, the prognosis for this disease is still very poor, because most types of HCC are resistant to conventional chemotherapeutic agents and have high recurrence after resection with curative aim. Currently, the only chemotherapeutic agent for HCC is sorafenib, however, it just expands the survival only 2.8 months [
4]. Therefore, the novel target and agents for HCC is unmet need.
Cancer stem cells (CSCs) are identified by experiments in which tumor cells are fractionated, characterized by cell surface markers, and injected at limiting dilutions into mice. Those populations that lead to tumor growth in the animal, and that lead to tumor growth when that tumor is subsequently transplanted into a second animal, are considered as CSCs [
5]. Studies have shown that CSCs can be resistant to common forms of cancer treatment such as chemo- and radiation therapy, resulting in tumor recurrence, metastasis, and treatment failure [
6‐
8]. Therefore, deeper knowledge of the interactions between cancer cells and CSCs are needed to fully understand tumor development, progression, and chemo-resistance in HCC. Recently, compelling evidence has reported that HCC is hierarchically organized and originates from a primitive stem/progenitor [
9].
In particular, CD133 has drawn significant attention as an important liver CSC marker. CD133 was the first identified member of the prominin family of pentaspan transmembrane (5-transmembrane) glycoproteins. It is also commonly known in humans and rodents as Prominin 1 (PROM1) [
10]. In HCC, CD133-positive cells exhibit liver CSC-like properties, such as high clonogenicity, tumorigenicity, and resistance to radiation [
11,
12]
. Other studies have shown that the presence of CD133-positive cells in HCC patients after surgery is correlated with early recurrence and poor prognosis [
13,
14]
. However, despite of extensive research efforts, the specific signaling pathway and mechanism of action by which CD133-positive cells are able to evade conventional therapies in HCC or other cancer types remain largely unknown.
Reactive oxygen species (ROS), which are formed by the capture of electrons by an oxygen atom, are chemically reactive molecules that have essential functions in living organisms [
15]. In normal cells, moderate levels of ROS are essential for cellular proliferation, differentiation, and survival [
16,
17]. On the other hand, chronically increased endogenous ROS levels lead to adaptive changes that play pivotal roles in tumorigenesis, metastasis, and drug resistance in diverse types of cancer cells. Some anti-cancer drugs that increase ROS generation or inhibit ROS elimination can induce a significant accumulation of ROS in cancer cells, leading to oxidative damage and cell death [
18]. In recent times, the regulation of ROS levels in CSCs has emerged as an active field of research. CSCs have lower levels of intracellular ROS than do non-CSCs, possibly due to the increased expression of free radical scavenging systems [
19‐
21]. Studies have showed that specific molecules associated with CSCs negatively regulate ROS levels, with a resultant increase in stemness. CD44 is one such molecule that has been associated with CSCs in several types of tumors, promotes ROS resistance by interacting with and stabilizing the cystine/glutamate transporter xCT in human gastrointestinal cancer, and increased CD13 expression reduces ROS levels, promoting the survival of liver cancer stem cells via an epithelial-mesenchymal transition-like phenomenon [
22,
23]. However, the roles of CD133 in ROS regulation have not been reported.
In this paper, we show that CD133-positive HCC cells exhibit strong resistance to reactive oxygen species (ROS) via upregulation of glutathione (GSH) levels, and thereby play a central role in resistance to liver cancer therapy. Based on this functional roles of CD133, we also found that sulfasalazine specially modulates the redox status in CD133-positive HCC, and could thereby sensitize CD133-positive HCC to chemotherapeutic treatment. Our results suggest that the combination of sulfasalazine and conventional chemotherapy could potentially be an effective therapeutic strategy against CD133-positive HCC.
Methods
Cell culture
Huh7, Hep3B, PLC/PRF/5 and HepG2 cells (human HCC lines) were obtained from the Korean Cell Line Bank. Human HCC cell line Huh6 was kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Germany) and Fa2N-4 cells (human immortalized hepatocyte cell line) were purchased from Xenotech (Lenexa, KS, USA). HCC cell lines were cultured in Dulbecco’s minimal essential medium (DMEM; Welgene, Korea, LM001-05) supplemented with heat-inactivated 10% fetal bovine serum (FBS; Gibco, Gaitherburg, MD, USA) and 100U/ml Penicillin and 100 μg/ml Streptomycin (Gibco) at humidified 37 °C incubator under 5% CO2. Fa2N-4 cells were plated in collagen-coated plates. After cell attachment (approximately 3 ~ 6 h), serum-containing plating medium (XenoTech, K4000) was replaced with supporting culture medium (XenoTech, K4100.X).
Primary cell culture
HCC tissue was cut into 3-mm3 pieces and washed with 4 °C Hank’s balanced salt solution (Lonza, Basel, Switzerland) supplemented with 1× antibiotic-antimycotic (A/A) solution (Sigma-Aldrich, St Louis, MO, USA) and 1× penicillin-streptomycin (P/S) (Lonza) in 100-mm Petri dishes. After three washes with DMEM/nutrient mixture F-12 (DMEM/F12; Gibco) supplemented with 10% FBS, 1× A/A solution, and 1× P/S, the cells were resuspended in 10 ml of the same solution and incubated at 4 °C for 16 h. Next, tissue was washed with fresh DMEM/F12 and incubated with 2 ml of 2× collagenase II (BD Biosciences, Franklin Lakes, NJ, USA) at 37 °C in a shaking chamber for 90 min. After incubation, the tissues were washed with DMEM/F12 several times until the supernatant was clear. The pellet was resuspended in hepatocyte basal medium (Lonza, CC3199) containing 1× A/A solution, 10% FBS, and 5 μg/ml hepatocyte growth factor (R&D systems, Minneapolis, MN, USA) and plated on collagen type I-coated T-25 flasks (BD Biosciences) with 5×105cells.
Huh7 were seeded in a very low density on 100-mm dish (5x105cells/10 cm2). After attachment of cells, complete medium were changed to DMEM/F12 (Gibco, 10565–018) supplemented with 1 × B27 (Invitrogen, Eugene, OR, USA), 20 ng/ml basic fibroblast growth factor (Invitrogen), 20 ng/ml epidermal growth factor (EGF, Invitrogen), 25 μg/ml insulin (Sigma) (LCSC media). After cultivation for 7 ~ 10 days without changing the medium, floating spheroids were collected and moved to low-attach 6-well plate (Corning, NY, USA) for subculture or 384-well culture plate (Greiner Bio-one, Monroe, NC, USA) for immunostaining.
For the HCC spheroids culture, slowly pipette the 8 μl of Matrigel (BD bioscience) directly on surface, carefully spread to avoid bubbles, in 384-well culture plates and incubated at 37 °C until Matrigel was solidified. Trypsinized cells were centrifuged at 1200 rpm and resuspended in culture medium and plated onto the Matrigel coated plates at a density of 2 × 103cells/well. Cells were incubated for 30 min at 37 °C to settle to the Matrigel and slowly added 10% Matrigel-medium to the each wells. After maintaining for 5 days, Matrigel-medium was replaced every 2 days. For immunostaining, they were washed with 1 mM Glycine (Sigma) carefully, and then spheroid were moved to 384-well culture plate.
Detection of drug sensitivity in spheroids
For the drug sensitivity with or without pretreating of sulfasalazine in spheroids, LCSC spheroids and HCC spheroids were transferred to 96-well plate. Spheroid were treated with anti-cancer drug for 6 ~ 8 days, and spheroids were examined their size using Operetta® High Content Screening (HCS) System using × 10 objective in bright field. For SASP pretreating study, spheroid was treated with 200 μM sulfasalazine for 24 h before anti-cancer drug treatment.
Reagent and antibodies
Hoechst 33342 (H3570, 1:500), 2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA. C6827, 1:1000), ThiolTracker™ violet (T10095, 1:500), Alexa Fluor® 633 Phalloidin (A22284, 1:100), Goat anti-mouse Alexa Fluor® 488 (A11001, 1:500), Goat anti-rabbit Alexa Fluor® 633 (A21070, 1:500), Goat anti-mouse Alexa Fluor® 633 (A21050, 1:500) and Goat anti-rabbit Alexa Fluor® 488 (A11008, 1:500) were purchased from Molecular Probes (Invitrogen). Rabbit monoclonal anti-CD44 (EPR10133Y clone, ab51037, 1:1000) rabbit polyclonal anti-EpCAM (ab71916, 1:1000) and mouse monoclonal anti-CD90 (ab133350, 1:1000) were purchased from Abcam (CSP, Cambridge, England). Methotrexate, Doxorubicin, Cisplatin, Sorafenib, Sulfasalazine (SASP), Buthionine sulphoximine (BSO), Arsenic trioxide, Etoposide and Hydrogen peroxide (H2O2) were purchased from Sigma Chemicals. Mouse monoclonal anti-CD133/1 (AC133, 130-090-422, 1:100) was purchased from Miltenyi biotec (Bergisch Gladbach, Germany). Rabbit polyclonal anti-AFP (Dako, Denmark A/S, Denmark, A000829. 1:500) and mouse monoclonal anti-β-actin (Sigma, A5441, 1:10,000) antibodies were purchased from each of the indicated companies.
HCS imaging assay technology
Cells were seeded at a 2,500 cells/well density for 72 h incubation and 1,500cells/well density for 96 h incubation in 384-well plate (90% of confluence in analyzing day). After being treated with the indicated concentrations of various drugs for proper time, cells were washed with Dulbecco’s phosphate buffered saline (DPBS; Welgene) and stained with fluorescent probes or antibody. Automated live-cell multispectral image acquisition was performed on the Operetta® HCS System using × 20 objective (Perkin Elmer, Waltham, MA, USA). The fluorescence images were captured according to the optimal excitation and emission wavelengths of each probe. To capture enough cells (>100) for analysis, five image fields starting at the center of well were collected from each well. Image analysis was performed using the Image Mining software. A series of measurements from the nuclei, ROS, and ThiolTracker™ channel images were obtained for each drugs.
siRNA transfection
siRNA probes were designed by and purchased from Dharmacon (Lafayette, CO, USA). Huh7 cells were seeded with 1x106cells/10 cm2 and the medium was replaced with Opti-MEM (Gibco) when the cell density reached 40–50%. The sequences of siCD133 was as follows: CD133 #1, 5′-GCUAAGUACUAUCGUCGAA-3′; CD133 #2, 5′-GAACAAGUUUACAGUGACU-3′; CD133 #3, 5′-GAAGUAUGGGAGAACAAUA-3′; CD133 #4, 5′-UCACAAUCCUGUUAUGACA-3′; Cells were co-transfected with the four siRNAs targeting CD133 (siCD133) scramble (siCont) for 24 h using Lipofectamine® 2000 (Invitrogen).
Cell sorting
Huh7 cells were analyzed by fluorescence-activating cell sorting (FACS; BD bioscience). The cells were harvested using 0.05% trypsin (Gibco), washed twice with DPBS supplemented with 5% FBS and resuspended in DPBS supplemented with 10% FBS with mouse anti-human CD133/1 antibody (Miltenyi Biotec) for 30 min at 4 °C. The cells were washed twice with pre-cooled DPBS and centrifuged at 1200 rpm at 4 °C and incubated in DPBS supplemented with 10% FBS with goat anti-mouse Alexa Fluor® 488 for 30 min at 4 °C in dark. After washing with DPBS twice, cells were sorted by FACS. CD133−negative and CD133-positive HCC were collected for further experiments, and they were cultured in DMEM supplemented with 10% FBS with 1% of penicillin/streptomycin.
Flow cytometry analysis
For investigating the CD133 population after CD133 positive cell sorting or transfected with siCD133, cells were trypsinized and washed twice with DPBS supplemented with 5% FBS and resuspended in DPBS supplemented with 10% FBS with mouse anti-human CD133/1 antibody for 30 min at 4 °C. The cells were washed with DPBS and centrifuged at 1200 rpm a 4 °C and incubated n DPBS supplemented with 10% FBS with goat anti-mouse Alexa Fluor® 488 for 30 min at 4 °C in dark. After washing with DPBS twice, cells were analyzed by flow cytometry.
For analyzing of cellular ROS levels, the cells were incubated with 10 mM of CM-H2DCFDA at 37 °C for 10 min in the dark, and washed with DPBS. For detecting the GSH levels, the cells were washed with DPBS containing the Ca2+ and Mg2+ and then cells were treated with 20 mM ThiolTracker™ in DBPS containing the Ca2+ and Mg2+ at 37 °C incubator for 30 min in the dark. Washed cells were then trypsinized and suspended for flow cytometry, intensity of 405 nm were measured for detecting GSH level.
Analysis of total glutathione (GSH) activity
Assay for total GSH activity was performed using assay kit (Sigma) according to the manufacturer’s protocol. The method is based on the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) reaction and the products were read at 412 nm.
Polyacrylamide gel electrophoresis (PAGE) and western blot analysis
Cells were solubilized in lysis buffer (3 M, Maplewood, MN, USA), the samples were boiled for 5 min, and equal amounts of protein (10–30 μg/well) were separated on 8 or 10% SDS-PAGE gels. After electrophoresis, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and blocked with 5% skim milk for 30 min at R.T. After blocking, the PVDF membranes were incubated with anti-CD133, xCT and β-actin for 16 h at 4 °C. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at a 1:10000 dilution, and specific bands were visualized by enhanced chemiluminescence (ECL; Thermo Scientific) and recorded on X-Omat AR films (Eastman Kodak Co., Rochester, NY, USA).
Irradiation
Cells were plated in 100-mm dish and 6-well plate for each experiment, and tumor-bared mice were treated with 1Gy, 2Gy, 4Gy (for colony forming assay), 5Gy (xenografted mice), 10 Gy (cell survival, ROS accumulation) of ioning radiation (IR) using a 6 MV photon beam linear accelerator (CL/1800, Varian Medical System Inc., Palo Alto, CA, USA) [
24,
25].
Clonogenic survival assays
Briefly, Huh7-siCont and Huh7-siCD133 cells were seeded into 6-well plates (Nunc, Roskilde, Denmark) at a density of 500 cells/well and allowed to grow for 24 h. The cells were then treated with SASP (100 μM or 200 μM) for further 24 h [
26]. The SASP-containing media were then discarded, and the cells were washed with DPBS and culture to form colonies in complete medium after irradiation (1, 2, 4Gy).
Tumor xenografts in nude mice
Huh7 (5x10
6cells) with 95% viability were injected subcutaneously into the hind legs of 6-week-old BALB/c athymic nude mice (SLC Inc., Hamamatsu, Japan) [
27,
28]. When tumors reached a volume of 200–250 mm
3, mice were randomly allocated to four groups as follows: (1) the tumor control group, (2) the SASP group (5 mg/20 g, 9 days) [
22], (3) the IR group (5Gy), and (4) the SASP plus IR group. Each group contained three mice. Tumor volumes were determined using the following formula: (L × I
2)/2, where L = tumor length and I = tumor width. Dimensions were determined using calipers. Local tumor irradiation was performed under anesthesia using a 6 MV photon beam linear accelerator (CL/1800). SASP was dissolved in saline (0.9% NaCl) and injected for intraperitoneal for 9 days. The mice were sacrificed for immunohistochemistry, RT-PCR and western blot after 15 days from SASP injection.
Statistical analysis
All experiments were performed at least three times. The results are expressed as the mean ± SD. Statistical analysis was performed using the Student’s t-test.
Discussion
HCC is one of the most malignant human cancers, with high mortality rates worldwide in spite of early detection and improvements in therapeutic technology. Nowadays, surgical resection is considered as a first-line therapy for HCC, whereas systemic chemotherapy plays an integral role for patients with advanced HCC for whom surgery is not a feasible option [
29]. However, the effects of chemotherapeutics such as sorafenib [
30] and cisplatin [
31] on advanced HCC are extremely limited, because most types of HCC inherently possess drug resistance to chemotherapy [
32]. CSCs are considered the ‘Achilles heel’ of anticancer efforts, due to their strong resistance to chemotherapy and radiotherapy. Recent studies indicate that HCC progression and drug resistance might be derived from CSCs [
33]. Papers have shown that CSC-related surface markers and pathways can modulate tumor development and suppression in liver cancer [
9,
34‐
36]. Although the existence of CSCs in solid human tumors is widely accepted, details of their origin and the source of their chemoresistance are unclear [
37]. In reality, the culture and functional study of CSCs are difficult in vitro, because CSC enrichment is rapidly lost in artificial culture systems [
38]. In order to overcome this, we applied an alternative approach to enrich the CSC population by manipulating 3D culture conditions. From these attempts, we discovered that characteristics and population of CSCs are controlled by changes in the tumor microenvironment, and that CD133-positive HCC cells have CSC-like properties to maintain tumor survival from anti-cancer therapies (Fig.
1). CD133/Prominin-1 has attracted considerable attention as a representative liver CSC marker. Indeed, liver cancer patients with high CD133 expression levels were found to have shorter overall survival and higher recurrence rates than patients with low CD133 expression (13). Studies have shown that CD133-positive liver CSCs can induce aberrant signaling pathways different from those in CD133-negative cells, such as the Akt/PKB pathway, JNK, mTOR, ERK, and β-catenin
etc. [
9,
39,
40]. However, the specific mechanism of action by which CD133 CSCs are able to avoid conventional therapies in HCC remains unknown. Here, we revealed that HCC cells with high CD133 expression levels have a strong capacity for ROS defense compared to HCC cells with low levels of CD133 expression (Figs.
2,
3 and
4). Although the mechanisms regulating the expression of CD133 in hypoxic conditions are known [
41], the detailed mechanism by which CD133 expression is upregulated in response to oxidative stress has not been elucidated. Here, our data showed that CD133 expression is increased in HCC in response to oxidative stress (Fig.
2e, f).
Recently, the correlation between ROS status and chemo- and radio-resistance in CSCs has been revealed in diverse cancers. A subset of CSCs exhibits enhanced ROS defense compared to non-tumorigenic cells in breast tumors [
19] and lower intracellular concentrations of ROS and ATP can be used as indicators of CSCs in lung cancer [
20]. However, the mechanisms by which CSCs maintain lower levels of ROS in HCC remain hitherto unknown.
In the present study, we have found that CD133-positive HCC cells control intracellular ROS level via the upregulating of GSH and sulfasalazine (SASP) not only alleviates ROS defense capacity but also increases the therapeutic efficacy of conventional anticancer therapy in CD133-positive HCC cells but not in CD133-negative HCC cells in vivo and in vitro.
Actually, this kind of mechanism was proposed by Ishimoto et al. for another stem-like protein, CD44 [
22]. They demonstrated that ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in gastric cancer. Here, we examined a potential cross-talk between CD133 and CD44 on ROS status (Fig.
3b) and we found that CD133-positive cells perform defense against ROS with proposed mechanism which is indifferent of CD44 expression in HCC.
In this study, we pre-treated sulfasalazine (SASP) before treating anti-cancer drugs or radiation. We would like to emphasize the SASP with sensitizer for increasing the drug efficiency through increasing the ROS accumulation and decreasing the GSH. Additionally, we could hypothesis that CD133 inhibits ROS resistance through the maintenance of ROS-induced increasing xCT expression in CD133-positive HCC cells, and thereby plays a central role in resistance to liver cancer therapy. xCT inhibition by treatment with SASP could sensitize CD133-positive HCC cells to available anticancer therapies.
SASP not only alleviates ROS defense capacity but also increases the therapeutic efficacy of conventional anticancer therapy in CD133-positive HCC cells but not in CD133-negative HCC cells in vivo and in vitro (Figs.
5 and
6). To date, no single agent or combination therapy has demonstrated any advantage in terms of both overall survival and quality of life, representing an unmet need. Combination therapy has not improved overall survival but has nonetheless been in wide use for many years because of its possible roles in palliation. Thus, we herein suggest that combination therapy with SASP and existing anticancer therapies should be feasible for patients with HCC without imposing side effects, since SASP is already approved to treat rheumatoid arthritis without safety issues. Given that CD133-positive HCC cells play a central role in resistance to cancer therapy, we believe that selective inhibition of the CD133-positive HCC population by pretreatment with SASP should surpass the limitations of the existing treatment of liver cancer.