Abstract
Background
Osteosarcoma (OS) is a malignant bone tumor primarily affecting children and adolescents. The prognosis of chemotherapy-refractory OS patients is poor. We developed a tumor suppressor p53–expressing oncolytic adenovirus (OBP-702) that exhibits antitumor effects against human OS cells. Here, we demonstrate the chemosensitizing effect of OBP-702 in human OS cells.
Materials and methods
The in vitro and in vivo antitumor activities of doxorubicin (DOX) and OBP-702 were assessed using parental and DOX-resistant OS cells (U2OS, MNNG/HOS) and a DOX-resistant MNNG/HOS xenograft tumor model.
Results
DOX-resistant OS cells exhibited high multidrug resistant 1 (MDR1) expression, which was suppressed by OBP-702 or MDR1 siRNA, resulting in enhanced DOX-induced apoptosis. Compared to monotherapy, OBP-702 and DOX combination therapy significantly suppressed tumor growth in the DOX-resistant MNNG/HOS xenograft tumor model.
Conclusion
Our results suggest that MDR1 is an attractive therapeutic target for chemoresistant OS. Tumor-specific virotherapy is thus a promising strategy for reversing chemoresistance in OS patients via suppression of MDR1 expression.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Osteosarcoma (OS) is the most frequent primary malignant bone tumor in children and adolescents in the United States [1]. Owing to recent advances in multi-agent chemotherapy and surgical techniques, the prognosis of OS patients has gradually improved from 20 to 70% over the past three decades [2]. The most standard chemotherapy regimens for OS patients include doxorubicin (DOX), methotrexate, and cisplatin. However, the prognosis of OS patients who are refractory to chemotherapy remains poor due to local recurrence and distant metastasis [2]. Therefore, many clinical trials evaluating a variety of chemotherapy regimens have been conducted to improve the chemosensitivity of OS patients [3, 4]. The Children’s Cancer Group/Pediatric Oncology Group trial INT-0133 demonstrated that the combination of ifosfamide and/or muramyl tripeptide–phosphatidyl ethanolamine has not improved the outcome of OS patients treated with standard chemotherapy [5]. Since efforts to develop multimodal therapies to improve chemosensitivity in OS patients have been unsuccessful [6], chemoresistance remains the primary obstacle in the treatment of OS patients.
Multidrug resistance protein 1 (MDR1), also known as ATP-binding cassette sub-family B member 1 (ABCB1) or P-glycoprotein, reduces the intracellular concentration and cytotoxicity of chemotherapeutic drugs by pumping the drugs out of cells [7]. Overexpression of MDR1 protein has been demonstrated in many types of human cancers, including OS [8]. A number of studies have examined the inhibition of MDR1 expression using small interfering RNA (siRNA) as a means of overcoming chemoresistance in human cancer cells [9,10,11,12]. In OS patients treated with DOX–based chemotherapy, MDR1 overexpression at diagnosis is an important adverse prognostic factor for clinical outcome [13]. Therefore, although MDR1 is an attractive therapeutic target for modulating chemoresistance in cancer patients, the use of MDR1-targeting inhibitors has been unsuccessful in clinical settings [8].
Oncolytic virotherapy has recently emerged as a novel anticancer therapeutic strategy [14]. Tumor-specific replication-competent oncolytic viruses are promising antitumor reagents that induce tumor-specific cell death [15]. We developed a telomerase-specific replication-competent oncolytic adenovirus, designated OBP-301 (Telomelysin), which induces tumor-specific death in a variety of human cancer cells [16, 17]. A phase I clinical trial of OBP-301 in the United States demonstrated the safety of intratumoral injection in patients with various advanced solid tumors, including sarcomas [18]. We previously evaluated the antitumor effect of OBP-301 in human OS cells [19] and determined that OBP-301 enhances the antitumor effect of chemotherapeutic drugs in human OS cells [20]. However, as some OS tumors are relatively OBP-301 resistant [19], we recently developed a p53-armed telomerase-specific oncolytic adenovirus (OBP-702) that exhibits stronger antitumor effects compared to p53-free OBP-301 due to activation of the p53-mediated cell death signaling pathway [21, 22]. Therefore, OBP-702 is expected to be more effective than OBP-301 when combined with chemotherapy. However, the therapeutic potential of OBP-702 in combination with chemotherapy remains unclear.
In the present study, we investigated the therapeutic potential of OBP-702 as a chemosensitizing reagent against chemoresistant OS cells. The underlying mechanism of OBP-702–mediated chemosensitization was evaluated with respect to apoptosis and modulation of MDR1 protein expression. The in vivo antitumor effect of combination therapy was examined using a subcutaneous chemoresistant OS xenograft tumor model.
Materials and methods
Cell lines
Human U2OS cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Human MNNG/HOS cells were purchased from DS Pharma Biomedical (Osaka, Japan). Cells were cultured for no longer than 5 months following resuscitation. Authentification of cells was not performed by the authors. U2OS cells were maintained in McCoy’s 5a medium. MNNG/HOS cells were maintained in Eagle’s minimum essential medium containing 1% nonessential amino acids. All media were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were routinely maintained at 37 °C in a humidified atmosphere with 5% CO2.
Chemoresistant U2OS and MNNG/HOS cells were obtained by sequential exposure to DOX over 3 months according to methods described in a previous report [23]. The concentration of DOX was determined monthly based on 50% inhibitory concentration (IC50) values from XTT assays.
Recombinant adenoviruses
The telomerase-specific replication-competent adenovirus OBP-301 (Telomelysin), in which the promoter element of the hTERT gene drives expression of the E1A and E1B genes, was previously constructed and characterized [16, 17]. For OBP-301-mediated induction of exogenous p53 gene expression, we generated OBP-702, in which a human wild-type p53 gene expression cassette was inserted into the E3 region [21]. Recombinant adenoviruses were purified using cesium chloride step gradients, and virus titer was determined using a plaque-forming assay with 293 cells. Viruses were stored at − 80 °C.
Cell viability assay
Cells were seeded on 96-well plates at a density of 1 × 103 cells/well 24 h before administration of chemotherapeutic drugs or viral infection. For monotherapy, cells were treated with DOX at 0, 0.1, 1, 5, or 10 µg/mL or infected with OBP-702 at a multiplicity of infection (MOI) of 0, 1, 5, 10, 50, or 100 plaque-forming units (PFUs)/cell. For combination therapy involving chemotherapy and OBP-702 treatment, cells were infected with OBP-702 at the indicated MOIs. Two days after viral infection, cells were further treated with DOX for 24 h. This treatment protocol was established in our recent study [20] to induce a synergistic effect between the chemotherapeutic agent and oncolytic adenovirus. Cell viability was determined using a Cell Proliferation Kit II (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s protocol. The combination index was calculated using CalcuSyn software (BioSoft, Inc., Cambridge, UK).
Cell proliferation assay
Cells were seeded on 24-well plates at a density of 1 × 104 cells/well 24 h before treatment. Twenty-four hours later, cells were infected with OBP-702 at the indicated MOIs. Two days after viral infection, cells were treated with DOX at the indicated doses for 24 h. Cells were counted using a hemocytometer.
Western blot analysis
Cells were seeded in a 100-mm dish at a density of 2 × 105 cells/dish 24 h before treatment. The cells were then infected with OBP-702 or OBP-301 at the indicated MOIs or transfected with 10 nmol/L MDR1 siRNA or control siRNA (Applied Biosystems, Foster City, CA, USA). Two days after treatment, the cells were treated with DOX at the indicated doses for 24 h. Whole-cell lysates were prepared using lysis buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100] containing a protease inhibitor cocktail (Complete Mini; Roche). Proteins were electrophoresed on 6–15% SDS polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK). The membranes were blocked with Blocking-One (Nacalai Tesque, Kyoto, Japan) at room temperature for 30 min. The primary antibodies used were: mouse anti-Ad5 E1A monoclonal antibody (mAb) (BD PharMingen, Franklin Lakes, NJ, USA), rabbit anti-poly(ADP-ribose) polymerase (PARP) polyclonal antibody, rabbit anti-MDR1/ABCB1 mAb (Cell Signaling Technology, Danvers, MA, USA), and mouse anti–β-actin mAb (Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were: horseradish peroxidase–conjugated antibodies against rabbit IgG (GE Healthcare) and mouse IgG (GE Healthcare). Immunoreactive bands on the blots were visualized using enhanced chemiluminescence substrates (ECL Plus; GE Healthcare).
LC–MS analysis
Parental and DOX-resistant OS cells were lysed in lysis buffer [50 mM HEPES–NaOH (pH 8.0), 12 mM deoxycholate, 12 mM N-lauroyl sarcosinate] using low-intensity probe sonication. A total of 10 µg of protein was then reduced with 20 mM tris(2-carboxyethyl) phosphine (Sigma-Aldrich) at 100 °C for 10 min, followed by alkylation with 50 mM iodoacetamide (Sigma-Aldrich) at 25 °C in the dark for 45 min. Samples were mixed with Laemmli’s sample buffer and loaded onto a polyacrylamide SDS gel. Electrophoresis was stopped when the loading dye reached 2 mm beneath the top of the separating gel. The gel was stained with Coomassie Brilliant Blue, and protein bands were excised and subjected to in-gel digestion with 100 ng of Trypsin/Lys-C Mix (Promega, Madison, WI, USA) at 37 °C for 12 h. The resulting peptides were extracted from the gel fragments and analyzed using a LTQ Orbitrap-Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) connected to an UltiMate 3000 RSLC nano-flow HPLC system (DIONEX Corp., Sunnyvale, CA, USA). Protein identification and quantification were performed using MaxQuant software [24]. The MS/MS spectra were searched against the Homo sapiens protein database in SwissProt, with the false-discovery rate set to 1% for both the peptide and protein identification filters. Only “Razor + unique peptides” were used for calculation of relative protein concentration.
Quantitative real-time reverse transcription PCR analysis
To evaluate the expression of MDR1 mRNA, cells were seeded on 6-well plates at a density of 2 × 105 cells/well. Total RNA was extracted from the cells and tumor tissues using a miRNeasy Mini kit (Qiagen, Valencia, CA, USA). cDNA was synthesized from 10 ng of total RNA using a TaqMan reverse transcription kit (Applied Biosystems). MDR1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was assessed using quantitative real-time PCR with an Applied Biosystems StepOnePlus™ real-time PCR system (Applied Biosystems). Relative expression levels of MDR1 mRNA were calculated according to the 2−ΔΔCt method after normalization with reference to the expression of GAPDH mRNA.
Immunocytochemistry
To evaluate the subpopulation of MDR1-positive cells, cells were fixed with 4% paraformaldehyde for 15 min and were labeled with primary rabbit anti-MDR1 mAb (Cell Signaling Technology) at 4 °C overnight. Then cells were stained with secondary Alexafluor488 goat anti-rabbit IgG antibody (Invitrogen) for 30 min. DAPI was used for nuclear counterstaining. The photographs of immunostained cells were obtained under a fluorescence microscope (IX83; Olympus, Tokyo, Japan).
In vivo subcutaneous DOX-resistant MNNG/HOS xenograft tumor model
Animal experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Okayama University School of Medicine. DOX-resistant MNNG/HOS cells (5 × 106 cells/site) were inoculated subcutaneously into the right flank of 6-week-old female BALB/c-nu/nu mice (CLEA Japan, Tokyo, Japan). Palpable tumors developed within 21 days and were permitted to grow to approximately 5 to 6 mm in diameter. At that stage, a 100-µL volume of solution containing OBP-702 (5 × 107 PFUs) or PBS was injected into the tumors every week for 3 cycles. DOX (5 mg/kg) was injected intraperitoneally 2 days after OBP-702 injection for 3 cycles.
Histopathologic analysis
Tumors were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks. Sections were stained with hematoxylin/eosin to assess the tumor region. Proliferation and MDR1 expression of tumor cells within tumor tissues was assessed by immunostaining with rabbit anti-Ki67 mAb (Abcam, Cambridge, MA, USA) and rabbit anti-MDR1 mAb (Cell Signaling Technology), respectively, using standard techniques. All sections were analyzed under a light microscope. The number of immunoreactive cells for Ki67 or percentage of immunoreactive area for MDR1 were calculated in three randomly selected fields in each tumor using ImageJ software.
Statistical analysis
Data are expressed as the mean ± standard deviation (SD). The significance of differences was assessed using the Student’s t test. Differences between groups in animal experiments were assessed using one-way analysis of variance followed by Tukey’s multiple-group comparison test. Statistical significance was defined as a P value of < 0.05.
Results
Establishment of DOX-resistant OS cells
To analyze the therapeutic potential of OBP-702 against chemoresistant OS cells, we first established two types of DOX-resistant human OS cells (U2OS and MNNG/HOS) by sequential exposure to the standard chemotherapeutic agent DOX over 3 months. After establishment of chemoresistant OS cells, we compared the sensitivity of 4 human OS cell lines (parental U2OS, DOX-resistant U2OS, parental MNNG/HOS, and DOX-resistant MNNG/HOS) to DOX treatment. As expected, DOX-resistant U2OS and MNNG/HOS cells were significantly more resistant to DOX compared to parental cells (Fig. 1A). The DOX IC50 values for DOX-resistant U2OS and MNNG/HOS cells were approximately 20-fold higher than those of parental cells (Fig. 1B). Western blot analyses showed that DOX treatment induced apoptosis in a dose-dependent manner, which was confirmed by the increased expression of cleaved PARP, in parental U2OS and MNNG/HOS cells (Fig. 1C). By contrast, the expression of cleaved PARP was lower in DOX-resistant U2OS and MNNG/HOS cells than in parental cells with DOX treatment (Fig. 1C). The rate of DOX-resistant U2OS and MNNG/HOS cell proliferation was also lower than that of the parental cells (Supplementary Fig. S1); however, the morphology of DOX-resistant U2OS and MNNG/HOS cells was similar to that of the parental cells (Supplementary Fig. S2). These results indicate that DOX-resistant OS cells are less sensitive to DOX-induced apoptosis compared to parental cells.
Identification of chemoresistance-related proteins in DOX-resistant OS cells
To identify the molecular targets associated with chemoresistance in OS cells, we employed comprehensive and quantitative label-free proteomic analysis which finally provided relative expression levels of 2897 proteins in parental and DOX-resistant OS cells. A Venn diagram showed that 270 and 191 proteins were expressed at levels more than twofold higher in DOX-resistant U2OS and MNNG/HOS cells, respectively, and 97 proteins were commonly expressed at levels more than twofold higher in both DOX-resistant U2OS and MNNG/HOS cells when compared to parental cells (Fig. 2A and Supplementary Table S1). Among the 97 proteins that were expressed at levels more than twofold higher in DOX-resistant OS cells than parental cells, the top 5 included MDR1, PTHB1, A-kinase anchor protein SPHKAP, myosin regulatory light polypeptide 9, and epidermal growth factor receptor kinase substrate 8–like protein 2 (Fig. 2B). As MDR1 is a member of the ATP-binding cassette transporter family and associated with chemoresistance, we further confirmed that the expression level of MDR1 mRNA and protein was upregulated in DOX-resistant OS cells using quantitative real-time PCR and Western blot analyses, respectively (Fig. 2C, D). Immunocytochemistry for MDR1 demonstrated that the subpopulations of MDR1-positive cells were small and large in parental and DOX-resistant OS cells, respectively. These results suggest that MDR1-positive OS cells are selectively enriched under DOX treatment and targeting MDR1 activation holds therapeutic potential for overcoming chemoresistance in OS cells.
Chemosensitizing effect of OBP-702 in DOX-resistant OS cells
We recently confirmed the chemosensitizing effect of OBP-301 against various types of human malignant tumor cells [25], including OS cells [20]. To investigate the chemosensitizing effect of OBP-702 against human OS cells, we treated parental and DOX-resistant OS cells with DOX and OBP-702 as combination therapy. The number and viability of cells treated with the combination of OBP-702 and DOX decreased in a dose-dependent manner (Fig. 3A, B). Calculation of the combination index demonstrated a synergistic antitumor effect of combination therapy in parental and DOX-resistant OS cells (Fig. 3C). These results suggest that OBP-702 chemosensitizes both parental and DOX-resistant OS cells.
As parental OS cells were more sensitive to OBP-702 infection than DOX-resistant cells, we analyzed the expression level of coxsackie and adenovirus receptor (CAR) in parental and DOX-resistant OS cells by flow cytometric analysis. The expression of CAR protein was significantly higher in parental OS cells than DOX-treated cells (Supplementary Fig. S4). These results suggest that chemoresistance is associated with low CAR expression.
OBP-702–mediated suppression of MDR1 expression plays a critical role in enhancing DOX-induced apoptosis
To elucidate the underlying mechanism of OBP-702–mediated enhancement of chemosensitivity in DOX-resistant OS cells, we examined whether OBP-702 enhances DOX-mediated apoptosis. DOX-resistant U2OS and MNNG/HOS cells were treated with DOX and/or OBP-702, and then apoptosis status was assessed by Western blot analysis. In DOX-resistant U2OS and MNNG/HOS cells treated with OBP-702 alone, cleaved PARP expression increased and MDR1 expression decreased in a dose-dependent manner (Fig. 4A). To explore the mechanism of OBP-702-mediated MDR1 suppression, we analyzed whether OBP-301 without p53 induction downregulates MDR1 expression in DOX-resistant OS cells. Western blot analysis demonstrated that OBP-301 suppressed MDR1 expression in a dose-dependent manner as similar with OBP-702 (Fig. 4B). These results suggest that OBP-702-mediated MDR1 suppression is mainly due to adenoviral infection rather than p53 induction.
To investigate the role of OBP-702-mediated MDR1 suppression in DOX-resistant OS cells, we examined whether OBP-702 enhances DOX-induced apoptosis. In cells treated with DOX and OBP-702 in combination, MDR1 expression was suppressed, leading to enhanced DOX-induced apoptosis of DOX-resistant OS cells (Fig. 4C). To further confirm the role of MDR1 suppression in enhanced DOX-induced apoptosis, we assessed the effect of MDR1 knockdown by RNA interference in combination with DOX treatment. When MDR1 siRNA was combined with DOX, DOX-induced apoptosis was enhanced in DOX-resistant OS cells, similar to treatment with OBP-702 (Fig. 4D). These results suggest that suppression of MDR1 expression plays a crucial role in OBP-702–mediated enhancement of DOX-induced apoptosis.
In vivo antitumor effect of combination therapy with DOX and OBP-702
To assess the in vivo antitumor effect of combination therapy with DOX and OBP-702, we used a subcutaneous DOX-resistant MNNG/HOS xenograft tumor model. We injected OBP-702 or PBS into tumors weekly, and DOX was injected intraperitoneally 2 days after OBP-702 injection for 3 cycles (Fig. 5A). Combined treatment with DOX and OBP-702 significantly suppressed tumor growth when compared with mock treatment, whereas there was no significant difference between mock treatment and DOX or OBP-702 monotherapy (Fig. 5A, B). In the tumor growth curves of individual mice bearing DOX-resistant MNNG/HOS tumors (Supplementary Fig. S4), combination therapy completely suppressed tumor growth in one mouse. Histopathologic analysis for Ki67 and MDR1 revealed large necrotic areas lacking Ki67 and MDR1 expression in tumors treated with combination therapy (Fig. 5C). Combination treatment with OBP-702 significantly decreased the number of Ki67-positive cells and percentage of MDR1 expression area in DOX-treated OS tumors (Fig. 5D). These results suggest that OBP-702 enhances DOX-associated antitumor effects against DOX-resistant OS tumors via suppression of MDR1 expression.
Discussion
The prognosis of chemotherapy-refractory OS patients is typically poor, despite the recent advances in multi-agent chemotherapy and surgical resection therapies. Chemoresistance is thus the primary obstacle to improving clinical outcomes in OS patients. In this study, we demonstrated that combination therapy with DOX and OBP-702 induces a synergistic antitumor effect in parental and DOX-resistant OS cells. DOX-resistant OS cells exhibited higher MDR1 expression than parental OS cells. OBP-702 efficiently promoted the DOX-induced apoptosis of DOX-resistant OS cells by suppressing the expression of MDR1 as effectively as MDR1 siRNA. Moreover, combination therapy with OBP-702 and DOX significantly suppressed tumor growth in a subcutaneous DOX-resistant OS xenograft tumor model. Thus, OBP-702 is a promising antitumor reagent for modulating chemoresistance in OS cells via suppression of MDR1 expression.
Our proteomics analysis demonstrated that MDR1 protein was one of 97 proteins expressed at greater than twofold higher levels in DOX-resistant U2OS and MNNG/HOS cells than parental cells. The expression of MDR1 mRNA was also upregulated in DOX-resistant OS cells and tumors. Consistent with our findings, several reports have demonstrated the importance of MDR1 activation in the chemoresistance of OS cells [23] and OS tissues [26, 27]. Regarding the underlying mechanism of chemotherapy-associated MDR1 activation, interplay between genetic and epigenetic effects is thought to induce MDR1 expression in chemoresistant OS cells following chemotherapy [28]. Activation of the MDR1 gene is associated with the selection of spontaneous mutations and acetylation of histone H3 within the MDR1 upstream promoter. Liu et al. [29] suggested that disruption of the MDR1 gene via genome editing using the clustered regulatory interspaced short palindrome repeats–associated Cas9 system is associated with reversal of chemoresistance in OS cells. Thus, inhibition of MDR1 expression appears to be a promising therapeutic strategy for modulating chemoresistance in OS cells.
Various MDR1-targeting inhibitors have been developed to overcome chemoresistance in human cancers [8, 30]. However, the effect of oncolytic virotherapy on MDR1 expression in chemoresistant OS cells remains unclear. In this study, we demonstrated that OBP-702 suppresses MDR1 expression and subsequent enhancement of DOX-induced apoptosis. As OBP-301 without p53 induction also suppressed MDR1 expression, at least two possible mechanisms induced by adenoviral infection could govern OBP-702–mediated MDR1 suppression. One mechanism involves adenoviral E1A–dependent suppression of MDR1 expression. E1A protein has demonstrated therapeutic potential for enhancing the chemosensitivity of various human tumor cells [31]. Mantwill et al. [32] reported that E1A plays a critical role in adenovirus-mediated suppression of MDR1 expression. The second potential mechanism involves c-Jun NH2-terminal kinase (JNK)-dependent suppression of MDR1 expression. Adenoviral infection has been shown to induce JNK activation in the infected cells [33]. Zhou et al. [34] demonstrated that JNK represses transcription of the MDR1 gene. Taken together, these data suggest that OBP-702–mediated activation of E1A and JNK protein cooperatively downregulates MDR1 expression in DOX-resistant OS cells.
MDR1 protein is expressed in normal tissues as well as tumor tissues [8], suggesting that the use of MDR1-targeting inhibitors could have adverse effects [30]. MDR1 protein is expressed in hematopoietic stem cells [35] and various immune cells [36], including dendritic cells, NK cells, and CD8+ T cells. Indeed, MDR1-targeting inhibitors have been shown to attenuate the maturation of dendritic cells [37] and the cytotoxic function of NK cells [38] and CD8+ T cells [39]. Therefore, systemic administration of MDR1-targeting inhibitors could adversely affect antitumor immunity, although such inhibitors could enhance chemosensitivity. By contrast, oncolytic virotherapy is a promising antitumor strategy for specifically targeting tumor cells without affecting normal cells [14, 15]. Recent evidence suggests that oncolytic virotherapy enhances antitumor immunity by inducing the release of damage-associated molecular pattern molecules and tumor-associated antigens [40, 41]. Therefore, to overcome chemoresistance without adversely affecting antitumor immunity, OBP-702–mediated tumor-specific suppression of MDR1 expression may be more beneficial than MDR1-targeting inhibitors.
It is worth noting that DOX-resistant OS cells show the low proliferative ability, which is thought to be one of the characteristics for cancer stem-like cells. DOX treatment mainly targets proliferating cancer cells by inducing the intercalation into DNA base pairs, nucleosome destabilization, breakage of DNA strands, and inhibition of both DNA and RNA synthesis [42]. Dormant cancer stem-like cells would resist the DOX-mediated cytotoxic effect. In contrast, DOX-resistant OS cells showed the low CAR expression. CAR expression has been shown to be associated with cell cycle status, in which CAR expression is higher in the M phase than in the G1 and G2 phases [43]. Dormant cancer stem-like cells may be less sensitive to adenovirus infection than cycling cancer cells. Previous report has shown that DOX treatment induces chemoresistance with stem-like phenotype in U2OS and MNNG/HOS cells [44]. As we previously demonstrated that OBP-301 efficiently eliminates gastric cancer stem-like cells with chemoradioresistant phenotype by suppressing their dormancy [45], further experiments would be warranted to evaluate whether OBP-702 shows the therapeutic potential against dormant OS stem-like cells.
In conclusion, we demonstrated that the oncolytic adenovirus OBP-702 enhances the DOX-induced apoptosis of human OS cells via suppression of MDR1 expression, which is a critical factor in the chemoresistance of OS cells. Thus, combination therapy with DOX and OBP-702 is a novel and promising strategy for overcoming chemoresistance in OS patients. To investigate the tolerability and efficacy of combination therapy with OBP-702 and chemotherapy, further clinical studies are warranted in chemotherapy-refractory OS patients.
Abbreviations
- OS:
-
Osteosarcoma
- DOX:
-
Doxorubicin
- MDR1:
-
Multidrug resistance 1
- ABCB1:
-
ATP-binding cassette sub-family B member 1
- IC50 :
-
50% Inhibitory concentration
- MOI:
-
Multiplicity of infection
- PFU:
-
Plaque-forming unit
- mAb:
-
Monoclonal antibody
- PARP:
-
Poly(ADP-ribose) polymerase
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- CAR:
-
Coxsackie and adenovirus receptor
- JNK:
-
C-Jun NH2-terminal kinase
References
Siegel RL, Miller KD, Jemal A (2018) Cancer statistics. CA Cancer J Clin 68(1):7–30. https://doi.org/10.3322/caac.21442
Lamplot JD, Denduluri S, Qin J, Li R, Liu X, Zhang H, Chen X, Wang N, Pratt A, Shui W, Luo X, Nan G, Deng ZL, Luo J, Haydon RC, He TC, Luu HH (2013) The current and future therapies for human osteosarcoma. Curr Cancer Ther Rev 9(1):55–77. https://doi.org/10.2174/1573394711309010006
Isakoff MS, Bielack SS, Meltzer P, Gorlick R (2015) Osteosarcoma: current treatment and a collaborative pathway to success. J Clin Oncol 33(27):3029–3035. https://doi.org/10.1200/JCO.2014.59.4895
Gorlick R, Janeway K, Lessnick S, Randall RL, Marina N (2013) Children’s Oncology Group’s 2013 blueprint for research: bone tumors. Pediatr Blood Cancer 60(6):1009–1015. https://doi.org/10.1002/pbc.24429
Meyers PA, Schwartz CL, Krailo MD, Healey JH, Bernstein ML, Betcher D, Ferguson WS, Gebhardt MC, Goorin AM, Harris M, Kleinerman E, Link MP, Nadel H, Nieder M, Siegal GP, Weiner MA, Wells RJ, Womer RB, Grier HE (2008) Osteosarcoma: the addition of muramyl tripeptide to chemotherapy improves overall survival—a report from the Children’s Oncology Group. J Clin Oncol 26(4):633–638. https://doi.org/10.1200/JCO.2008.14.0095
Bishop MW, Janeway KA, Gorlick R (2016) Future directions in the treatment of osteosarcoma. Curr Opin Pediatr 28(1):26–33. https://doi.org/10.1097/MOP.0000000000000298
Dean M, Rzhetsky A, Allikmets R (2001) The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 11(7):1156–1166. https://doi.org/10.1101/gr.184901
Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2(1):48–58. https://doi.org/10.1038/nrc706
Wu H, Hait WN, Yang JM (2003) Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells. Cancer Res 63(7):1515–1519
Nieth C, Priebsch A, Stege A, Lage H (2003) Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi). FEBS Lett 545(2–3):144–150
Yague E, Higgins CF, Raguz S (2004) Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1. Gene Ther 11(14):1170–1174. https://doi.org/10.1038/sj.gt.3302269
Pichler A, Zelcer N, Prior JL, Kuil AJ, Piwnica-Worms D (2005) In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein. Clin Cancer Res 11(12):4487–4494. https://doi.org/10.1158/1078-0432.CCR-05-0038
Serra M, Pasello M, Manara MC, Scotlandi K, Ferrari S, Bertoni F, Mercuri M, Alvegard TA, Picci P, Bacci G, Smeland S (2006) May P-glycoprotein status be used to stratify high-grade osteosarcoma patients? Results from the Italian/Scandinavian Sarcoma Group 1 treatment protocol. Int J Oncol 29(6):1459–1468
Russell SJ, Peng KW, Bell JC (2012) Oncolytic virotherapy. Nat Biotechnol 30(7):658–670. https://doi.org/10.1038/nbt.2287
Fujiwara T, Shirakawa Y, Kagawa S (2011) Telomerase-specific oncolytic virotherapy for human gastrointestinal cancer. Expert Rev Anticancer Ther 11(4):525–532. https://doi.org/10.1586/era.10.200
Kawashima T, Kagawa S, Kobayashi N, Shirakiya Y, Umeoka T, Teraishi F, Taki M, Kyo S, Tanaka N, Fujiwara T (2004) Telomerase-specific replication-selective virotherapy for human cancer. Clin Cancer Res 10(1 Pt 1):285–292
Hashimoto Y, Watanabe Y, Shirakiya Y, Uno F, Kagawa S, Kawamura H, Nagai K, Tanaka N, Kumon H, Urata Y, Fujiwara T (2008) Establishment of biological and pharmacokinetic assays of telomerase-specific replication-selective adenovirus. Cancer Sci 99(2):385–390. https://doi.org/10.1111/j.1349-7006.2007.00665.x
Nemunaitis J, Tong AW, Nemunaitis M, Senzer N, Phadke AP, Bedell C, Adams N, Zhang YA, Maples PB, Chen S, Pappen B, Burke J, Ichimaru D, Urata Y, Fujiwara T (2010) A phase I study of telomerase-specific replication competent oncolytic adenovirus (telomelysin) for various solid tumors. Mol Ther 18(2):429–434. https://doi.org/10.1038/mt.2009.262
Sasaki T, Tazawa H, Hasei J, Kunisada T, Yoshida A, Hashimoto Y, Yano S, Yoshida R, Uno F, Kagawa S, Morimoto Y, Urata Y, Ozaki T, Fujiwara T (2011) Preclinical evaluation of telomerase-specific oncolytic virotherapy for human bone and soft tissue sarcomas. Clin Cancer Res 17(7):1828–1838. https://doi.org/10.1158/1078-0432.CCR-10-2066
Osaki S, Tazawa H, Hasei J, Yamakawa Y, Omori T, Sugiu K, Komatsubara T, Fujiwara T, Sasaki T, Kunisada T, Yoshida A, Urata Y, Kagawa S, Ozaki T, Fujiwara T (2016) Ablation of MCL1 expression by virally induced microRNA-29 reverses chemoresistance in human osteosarcomas. Sci Rep 6:28953. https://doi.org/10.1038/srep28953
Yamasaki Y, Tazawa H, Hashimoto Y, Kojima T, Kuroda S, Yano S, Yoshida R, Uno F, Mizuguchi H, Ohtsuru A, Urata Y, Kagawa S, Fujiwara T (2012) A novel apoptotic mechanism of genetically engineered adenovirus-mediated tumour-specific p53 overexpression through E1A-dependent p21 and MDM2 suppression. Eur J Cancer 48(14):2282–2291. https://doi.org/10.1016/j.ejca.2011.12.020
Hasei J, Sasaki T, Tazawa H, Osaki S, Yamakawa Y, Kunisada T, Yoshida A, Hashimoto Y, Onishi T, Uno F, Kagawa S, Urata Y, Ozaki T, Fujiwara T (2013) Dual programmed cell death pathways induced by p53 transactivation overcome resistance to oncolytic adenovirus in human osteosarcoma cells. Mol Cancer Ther 12(3):314–325. https://doi.org/10.1158/1535-7163.MCT-12-0869
Serra M, Scotlandi K, Manara MC, Maurici D, Lollini PL, De Giovanni C, Toffoli G, Baldini N (1993) Establishment and characterization of multidrug-resistant human osteosarcoma cell lines. Anticancer Res 13(2):323–329
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372. https://doi.org/10.1038/nbt.1511
Fujiwara T, Kagawa S, Tazawa H (2012) Synergistic interaction of telomerase-specific oncolytic virotherapy and chemotherapeutic agents for human cancer. Curr Pharm Biotechnol 13(9):1809–1816
Serra M, Scotlandi K, Manara MC, Maurici D, Benini S, Sarti M, Campanacci M, Baldini N (1995) Analysis of P-glycoprotein expression in osteosarcoma. Eur J Cancer 31A(12):1998–2002
Baldini N, Scotlandi K, Barbanti-Brodano G, Manara MC, Maurici D, Bacci G, Bertoni F, Picci P, Sottili S, Campanacci M et al (1995) Expression of P-glycoprotein in high-grade osteosarcomas in relation to clinical outcome. N Engl J Med 333(21):1380–1385. https://doi.org/10.1056/NEJM199511233332103
Chen KG, Wang YC, Schaner ME, Francisco B, Duran GE, Juric D, Huff LM, Padilla-Nash H, Ried T, Fojo T, Sikic BI (2005) Genetic and epigenetic modeling of the origins of multidrug-resistant cells in a human sarcoma cell line. Cancer Res 65(20):9388–9397. https://doi.org/10.1158/0008-5472.CAN-04-4133
Liu T, Li Z, Zhang Q, De Amorim BK, Lozano-Calderon S, Choy E, Hornicek FJ, Duan Z (2016) Targeting ABCB1 (MDR1) in multi-drug resistant osteosarcoma cells using the CRISPR-Cas9 system to reverse drug resistance. Oncotarget 7(50):83502–83513. https://doi.org/10.18632/oncotarget.13148
Robey RW, Pluchino KM, Hall MD, Fojo AT, Bates SE, Gottesman MM (2018) Revisiting the role of ABC transporters in multidrug-resistant cancer. Nat Rev Cancer 18(7):452–464. https://doi.org/10.1038/s41568-018-0005-8
Chang YW, Hung MC, Su JL (2014) The anti-tumor activity of E1A and its implications in cancer therapy. Arch Immunol Ther Exp 62(3):195–204. https://doi.org/10.1007/s00005-014-0273-2
Mantwill K, Kohler-Vargas N, Bernshausen A, Bieler A, Lage H, Kaszubiak A, Surowiak P, Dravits T, Treiber U, Hartung R, Gansbacher B, Holm PS (2006) Inhibition of the multidrug-resistant phenotype by targeting YB-1 with a conditionally oncolytic adenovirus: implications for combinatorial treatment regimen with chemotherapeutic agents. Can Res 66(14):7195–7202. https://doi.org/10.1158/0008-5472.can-05-2339
Klein SR, Piya S, Lu Z, Xia Y, Alonso MM, White EJ, Wei J, Gomez-Manzano C, Jiang H, Fueyo J (2015) C-Jun N-terminal kinases are required for oncolytic adenovirus-mediated autophagy. Oncogene 34(41):5295–5301. https://doi.org/10.1038/onc.2014.452
Zhou J, Liu M, Aneja R, Chandra R, Lage H, Joshi HC (2006) Reversal of P-glycoprotein-mediated multidrug resistance in cancer cells by the c-Jun NH2-terminal kinase. Cancer Res 66(1):445–452. https://doi.org/10.1158/0008-5472.CAN-05-1779
Chaudhary PM, Roninson IB (1991) Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 66(1):85–94
Bossennec M, Di Roio A, Caux C, Menetrier-Caux C (2018) MDR1 in immunity: friend or foe? Oncoimmunology 7(12):e1499388. https://doi.org/10.1080/2162402X.2018.1499388
Pendse SS, Behjati S, Schatton T, Izawa A, Sayegh MH, Frank MH (2006) P-glycoprotein functions as a differentiation switch in antigen presenting cell maturation. Am J Transpl Off J Am Soc Transpl Am Soc Transpl Surg 6(12):2884–2893. https://doi.org/10.1111/j.1600-6143.2006.01561.x
Ludescher C, Pall G, Irschick EU, Gastl G (1998) Differential activity of P-glycoprotein in normal blood lymphocyte subsets. Br J Haematol 101(4):722–727
Gupta S, Kim CH, Tsuruo T, Gollapudi S (1992) Preferential expression and activity of multidrug resistance gene 1 product (P-glycoprotein), a functionally active efflux pump, in human CD8+ T cells: a role in cytotoxic effector function. J Clin Immunol 12(6):451–458
Chiocca EA, Rabkin SD (2014) Oncolytic viruses and their application to cancer immunotherapy. Cancer Immunol Res 2(4):295–300. https://doi.org/10.1158/2326-6066.CIR-14-0015
Tazawa H, Kuroda S, Hasei J, Kagawa S, Fujiwara T (2017) Impact of autophagy in oncolytic adenoviral therapy for cancer. Int J Mol Sci. https://doi.org/10.3390/ijms18071479
Yang F, Teves SS, Kemp CJ, Henikoff S (2014) Doxorubicin, DNA torsion, and chromatin dynamics. Biochim Biophys Acta 1845 1:84–89. https://doi.org/10.1016/j.bbcan.2013.12.002
Seidman MA, Hogan SM, Wendland RL, Worgall S, Crystal RG, Leopold PL (2001) Variation in adenovirus receptor expression and adenovirus vector-mediated transgene expression at defined stages of the cell cycle. Mol Ther 4(1):13–21. https://doi.org/10.1006/mthe.2001.0414
Martins-Neves SR, Paiva-Oliveira DI, Wijers-Koster PM, Abrunhosa AJ, Fontes-Ribeiro C, Bovee JV, Cleton-Jansen AM, Gomes CM (2016) Chemotherapy induces stemness in osteosarcoma cells through activation of Wnt/beta-catenin signaling. Cancer Lett 370(2):286–295. https://doi.org/10.1016/j.canlet.2015.11.013
Yano S, Tazawa H, Hashimoto Y, Shirakawa Y, Kuroda S, Nishizaki M, Kishimoto H, Uno F, Nagasaka T, Urata Y, Kagawa S, Hoffman RM, Fujiwara T (2013) A genetically engineered oncolytic adenovirus decoys and lethally traps quiescent cancer stem-like cells in S/G2/M phases. Clin Cancer Res 19(23):6495–6505. https://doi.org/10.1158/1078-0432.CCR-13-0742
Acknowledgements
We thank Mr. Takeshi Ieda, Ms. Tomoko Sueishi, and Ms. Tae Yamanishi for their excellent technical support.
Funding
This study was supported in part by grants from the Japan Agency for Medical Research and Development (T. Fujiwara, No. 17ck0106285h001) and grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (T. Fujiwara, nos. 25293283 and 16H05416; T. Ozaki, no. 25293323; T. Kunisada, nos. 25462333 and 16K10862; K. Sugiu, no. 15K10446; and H. Tazawa, no. 16K10596).
Author information
Authors and Affiliations
Contributions
Conception and design: HT, TO, TF. Development of methodology: KS, HT, JH, SO. Acquisition of data (provided animals, provided facilities, etc.): KS, YY, TO, TK, YM, HK, AY, KU. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): KS, HT, JH, TF, KU. Writing, review, and/or revision of the manuscript: KS, HT, JH, TF. Administrative, technical, or material support: YU. Study supervision: HT, TK, SK, TO, TF.
Corresponding author
Ethics declarations
Conflict of interest
Y. Urata is President and CEO of Oncolys BioPharma, Inc. H. Tazawa and T. Fujiwara are consultants of Oncolys BioPharma, Inc. The other authors have no potential conflicts of interest to disclose.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Sugiu, K., Tazawa, H., Hasei, J. et al. Oncolytic virotherapy reverses chemoresistance in osteosarcoma by suppressing MDR1 expression. Cancer Chemother Pharmacol 88, 513–524 (2021). https://doi.org/10.1007/s00280-021-04310-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00280-021-04310-5