Targeting Notch1 signaling pathway positively affects the sensitivity of osteosarcoma to cisplatin by regulating the expression and/or activity of Caspase family
© Wang et al.; licensee BioMed Central Ltd. 2014
Received: 14 January 2014
Accepted: 26 May 2014
Published: 3 June 2014
The introduction of cisplatin has improved the long-term survival rate in osteosarcoma patients. However, some patients are intrinsically resistant to cisplatin. This study reported that the activation of Notch1 is positively correlated with cisplatin sensitivity, evidenced by both clinical and in vitro data.
In this study, a total 8 osteosarcoma specimens were enrolled and divided into two groups according to their cancer chemotherapeutic drugs sensitivity examination results. The relationship between Notch1 expression and cisplatin sensitivity of osteosarcoma patients was detected by immunohistochemistry and semi-quantitative analysis. Subsequently, two typical osteosarcoma cell lines, Saos-2 and MG63, were selected to study the changes of cisplatin sensitivity by up-regulating (NICD1 plasmid transfeciton) or decreasing (gamma-secretase complex inhibitor DAPT) the activation state of Notch1 signaling pathway. Our results showed a significant correlation between the expression of Notch1 and cisplatin sensitivity in patient specimens. In vitro, Saos-2 with higher expression of Notch1 had significantly better cisplatin sensitivity than MG63 whose Notch1 level was relatively lower. By targeting regulation in vitro, the cisplatin sensitivity of Saos-2 and MG63 had significantly increased after the activation of Notch1 signaling pathway, and vice versa. Further mechanism investigation revealed that activation/inhibition of Notch1 sensitized/desensitized cisplatin-induced apoptosis, which probably depended on the changes in the activity of Caspase family, including Caspase 3, Caspase 8 and Caspase 9 in these cells.
Our data clearly demonstrated that Notch1 is critical for cisplatin sensitivity in osteosarcoma. It can be used as a molecular marker and regulator for cisplatin sensitivity in osteosarcoma patients.
Osteosarcoma is the most common primary malignant bone tumor. It has a typical age related incidence rate, with a first peak occurring in the second decade (3.1-4.2 per 1,000,000 person years) and a second peak occurring in patients older than sixty (3.3-4.6 per 1,000,000 person years) [1–3]. Current treatments for osteosarcoma include combined approaches of surgery dissection and systemic chemotherapy. It is undeniable that the introduction of adjuvant and neoadjuvant chemotherapies has improved the long-term survival rate impressively [4–6]. For example, the use of cisplatin has been proved to be a useful chemotherapeutic method for preoperative induction therapy for osteosarcoma. Good responders to preoperative cisplatin chemotherapy showed a better survival rate . Besides, it was also well summarized that the inclusion of cisplatin had better outcome for high grade osteosarcoma .
Cisplatin is an effective antitumor agent with a wide spectrum of activity against human solid tumors [9–13]. Generally, it can form bivalent adducts with nucleophilic sites on purines in DNA, yield predominantly DNA intra-strand cross links between adjacent purines , and thus exert antitumor effects. However, while some common tumors are sensitive to cisplatin treatment, others are instrinsically resistant to cisplatin , leading to failure in the curative therapy. However, the underlying mechanisms of cisplatin resistance are still unclear. Identifying molecules that contribute to cisplatin resistance would be necessary and significant for the optimization of curative effects.
The Notch signaling pathway is an evolutionarily conserved ligand-receptor signaling system that regulates cell proliferation, survival, apoptosis and differentiation [16, 17]. Four Notch receptors have been identified in mammals, namely Notch1-4. Upon the binding of a ligand (jagged 1, jagged 2, delta-like 1 or delta-like 4) to the cell surface Notch receptors (Notch1-4), the Notch intracellular domain (NICD) will be cleaved by the gamma-secretase complex and translocated into the nucleus to induce the expression of downstream targeting genes including Hes1, Hes5, Hes7, Hey1, Hey2 and HeyL etc.. Dysfunction of Notch signaling pathway may lead to anomalous differentiation or undifferentiation, ultimately causing these cells toward malignant transformation. Indeed, many observations suggested that alterations in Notch signaling are associated with many human cancers [19–23]. In addition, Notch signaling pathway is also involved in chemotherapy drug-resistance. For instance, Notch1 plays an important role in the mechanisms of cisplatin resistance in several malignant tumors, such as head and neck squamous cell tumors, colorectal tumors and ovarian cancer . However, the roles of Notch1 signaling in osteosarcoma and chemotherapy response have not been elucidated yet. Therefore, this study aims to determine the roles of Notch1 signaling pathway in osteosarcoma, as well as the mechanisms of cisplatin response in osteosarcoma.
Materials and methods
Patients and specimens
Conventional osteosarcoma patients involved in this study were hospitalized in the Department of Orthopedic Surgery of the Ninth People’s Hospital of Shanghai, China between September 2010 and March 2012. All patients who enrolled provided informed consent in accordance with our institution’s regulatory requirements, and we conducted the study according to guidelines of the ethic committee of Ninth People's Hospital of Shanghai Jiaotong University.
In all cases, diagnosis of conventional osteosarcoma was established by clinical characteristics, radiological findings and pathological examination. Participants were randomly selected from our sample library, included seven patients (six males, one female) with median age of 31.6 years (range: 17–58 years). However, it is worthwhile to explain that one of these patients suffered a tumor recurrence after surgery operation within one year. Therefore, we have a total of eight histological specimens: sample 001–008 (sample 003 and sample 005 were both from the recurrence patient) which were all obtained after lesion excision. Then according to the inhibition rate of cisplatin in the tumor susceptibility test results, the histological specimens of these patients were divided into two groups: cisplatin sensitive group (cisplatin sensitivity 50%-90%) and cisplatin insensitive group (cisplatin sensitivity 0-30%). The two groups had no significant difference in composition regarding age or sex (P > 0. 05).
Human osteosarcoma cell lines, Saos-2 and MG63 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). They were authenticated using DNA fingerprinting (variable number of tandem repeats), confirming that no cross-contamination occurred during this study. The cell lines were used within 6 months of resuscitation. Cells were grown in α-MEM (GIBCO, Grand Island, NY, USA) supplemented with 10% FBS (Hy-clone, Tauranga, New Zealand) and antibiotics (penicillin 100 U/ml, streptomycin 100 lg/ml; Hyclone, Logan, UT,USA) in 37°C humidified atmosphere with 5% CO2.
Immunohistochemistry and immunocytochemistry
Immunohistochemical staining for Notch1 and HES1 were performed on 5 μm thick formalin-fixed paraffin embedded sections. Deparafinization and rehydration were performed in xylene and ethanol solutions (reducing concentration 95%-70%). Sections were incubated in H2O2 solution (3% H2O2 in PBS buffer) for 30 minutes to block endogenous peroxydase. Antigen retrieval were performed in procedure with the retrieval buffer (PH = 9.0, TRIS 20 mmol/L, EDTA 0.05 mmol/L, 0.05% Tween 20) in 99°C bath for 20 minutes. Sections were then incubated with the primary antibody: anti-HES1 (diluted 1:250, Epitomics, USA) and anti-Notch1 (diluted 1:50, Epitomics, USA) at 4°C overnight. After rinsing with the PBS buffer, the secondary antibody (MaxVision TM HRP-Polymer anti-Rabbit IHC Kit, Maixin.Bio, China) was applied for 15 minutes at room temperature (RT). DAB (Maixin.Bio, China) solution was used for chromogen. At last, the Sections were counterstained with Hematoxylin (Sigma) to identify nuclei. Immunohistochemistry with the secondary antibody alone without primary antibody was carried out as a control. The images were acquired using a microscope (Leica DM 4000B) with BioQuant OSTEO II software (BioQuant Image AnalysisCorporation, Nashville, TN).
Immunocytochemistry for Notch1 of Saos-2 and MG63 cells were performed in 6-well plate. Supernatant was discarded and the plate was washed by PBS buffer for three times. Then 0.2% Triton X-100 was added to penetrate cytomembrane for 5 minutes and 5% BSA was used for 30 minutes to block unspecific binding of the antibodies. Subsequently, sections were incubated with the primary antibody against Notch1 (diluted 1:100, Epitomics, USA) at 4°C overnight. After washing three times with PBS buffer, the cells were incubated with anti-rabbit Alexa594 (1:500, Molecular Probes, USA) for 30 minutes. DAPI (300 nM, Molecular Probes, USA) was used to counterstain the nuclei. At last, the images were acquired using an inverted fluorescence microscope (IX71, Olympus, Japan) with DP Controller software.
Activation and inhibition of Notch1 signaling pathway of Saos-2 and MG63 cells
For the activation of Notch1 signaling pathway of Saos-2 and MG63 cells, the Notch1 intracellular domain (NICD-1) plasmid which was cloned 2.4 kb NICD-1 fragment to pcDNA3.1 vector using forward primer: 5′-CACC ATG GTG CTG CTG TCC CGC AA GCGCC and reverse primer: 5′- TGC TTT AAA TGC CAC AGG AAT GTG GG was used to transfect into the two cell lines according to the protocols of Lipofectamine™ Reagent (Invitrogen, USA). Three days later, we detected the expression of HES1 mRNA, the main downstream aim target of Notch signaling pathway, as the indication of the activation of Notch1 signaling pathway by the plasmid. Subsequently, these cells were seeded into 96-well plate for the follow-up studies.
LY-374973, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (ENZO Life Sciences, USA), the inhibitor of γ-secretase complex in Notch signaling pathway, was used to non-specific inhibit the Notch1 signaling pathway of Saos-2 and MG63 cells. The cells were incubated in DAPT (100 μmol/L) in the 6-well/96-well plates for 24 hours. And then DAPT was removed and the cells were washed with fresh medium twice before cisplatin added.
Total RNA was isolated by using the AxyPrepTM Multisource Total RNA Miniprepkit (Axygen, USA). An equivalent amount of RNA was converted into complementary DNA (cDNA) with PrimeScript™ RT reagent Kit (Takara,Japan). Subsequently, Real-time PCR was performed using an ABI 7500 Sequencing Detection System and SYBR®Premix Ex Taq (Takara, Japan). All of the procedures were performed according to the manufacturer’s protocols. Cycling condition was as follows: 40 cycles at 95°C for 5 seconds and 60°C for 34 seconds. The primer sequences are, for human Notch1: forward 5′-GGA GGC ATC CTA CCC TTT TC-3′, and reverse 5′-TGT GTT GCT GGA GCA TCT TC-3′; for human HES1: forward 5′-CTC TCT TCC CTC CGG ACT CT-3′ and reverse 5′-AGG CGC AAT CCA ATA TGA AC-3′; for human Caspase3: forward 5′-TTT TTC AGA GGG GAT CGT TG-3′ and reverse 5′-CGG CCT CCA CTG GTA TTT TA-3′; for human Caspase8: forward 5′-TGC AGG GTC TCA CTC TGT TG-3′ and reverse 5′-CAA AAA TCA GCC ATG TGT GG-3′; for human Caspase9: forward 5′-CTA GTT TGC CCA CAC CCA GT-3′; and for human GAPDH: forward 5′-CCT GCA CCA CCA ACT GCT TA-3′ and reverse 5′-AGG CCA TGC CAG TGA GCT T-3′. The comparative 2-ΔΔCT method was used to calculate the relative expression level of each target gene with GAPDH as the housekeeping gene.
At the end of incubation, cells were washed with PBS and dissolved in lysis buffer containing protease inhibitor. Equal amount of proteins in cell homogenates were subjected to 10% SDS–PAGE and transferred to 0.22 μm PVDF membranes. The membranes were blocked with 5% free fat milk at room temperature for 1 h and then incubated with primary antibodies (all in accordance with 1:1,000 dilution), including rabbit monoclonal anti-Notch1 intracellular domain, rabbit monoclonal anti- Pro-Caspase3/cleaved Caspase3, rabbit monoclonal anti-Caspase8 and mouse monoclonal Pro-Caspase9/cleaved Caspase9 (all were purchased from Cell Signaling Technology, USA), overnight at 4°C, after 3 washes in TBST, the membranes were incubated with anti-rabbit or anti-mouse IgG for 1 h at room temperature. After washing, the membranes were incubated with enhanced chemiluminescence system (ECL) detection kit (Amersham Life Science, Little Chalfont, UK). Positive immunoreactive bands were quantified densitometrically, normalized by GAPDH.
Remaining cells test after treated with cisplatin by CCK-8 assay
A cell count kit-8 (CCK-8, Dojindo, Japan) was employed in this experiment to quantitatively evaluate the remaining cells viability after Saos-2 and MG63 cells whose Notch1 signaling pathway was activated or inhibited were treated with cisplatin (10 μg/ml, 5 μg/ml and 2.5 μg/ml). Cisplatin application concentration was calculated according to the clinical treatment program (20 mg/m2 ≈ 5 μg/ml). Approximately 7 × 103 cells were seeded on each film placed in the 96-well plates. The culture medium was removed and the cells were washed with PBS before CCK-8 examination. Subsequently, 100 μl α-MEM medium and 10 μl CCK-8 solution were added to each sample, followed by incubation at 37°C for 2.5 hours. The optical density (OD) at 450 nm was determined using a microplate reader (BIO-TEK, USA). At last, all the CCK8 results in our study were divided by their self-control group to obtain the ratio of remaining cell and the proliferation rate: ;
Analysis of apoptosis
Cells were seeded at 200,000 cells per well in six-well plates. NICD-1 plasmid transfection cells were harvested at 24 hours after 5 μg/ml cisplatin treatment and 36 hours for DAPT treatment cells. Cells were washed twice with cold PBS and then re-centrifuged the washed cells, discarded the supernatants and resuspended the cells in 1X annexin-binding buffer. Early apoptosis was detected by staining with Alexa Fluor® 488 annexin V and Propidium iodide labeling using the Vybrant® Apoptosis Assay Kit #2 (Invitrogen, USA). FACS was performed using a FACScan flowcytometer (Beckman-Altra, USA). Data were acquired using CELL Quest software.
Each sample was analysed in triplicate, and experiments were repeated three times. Mean, standard deviation (SD), and P values base on the 2-tailed t test were calculated with Excel X (Microsoft). Differences were considered significant at P < 0.05.
Osteosarcoma patients with higher Notch1 expression are more sensitive to cisplatin treatment
Osteosarcoma cell line with higher Notch1 expression is more sensitive to cisplatin treatment
Activation of Notch1 signaling pathway sensitizes osteosarcoma cell lines to cisplatin treatment
Inhibition of Notch1 signaling pathway desensitizes osteosarcoma cell lines to cisplatin treatment
After targeted regulation of Notch1 signaling pathway, osteosarcoma cells apoptosis changes under the cisplatin effect
Inhibition of Notch1 signaling pathway decreases the expression and/or activity of Caspase3, Caspase8 and Caspase9 in osteosarcoma cells even under the effect of cisplatin
The use of multiagent, intensive chemotherapy has proved to be a major development in osteosarcoma treatment. It has dramatically changed osteosarcoma from a malignancy with a modest survival rate to one in which at least two thirds of patients could be cured [25, 26]. Cisplatin is one of the most common first-line chemotherapy drugs for osteosarcoma because of its DNA cross-linking activity . However, not all osteosarcoma patients are sensitive to cisplatin treatment . Therefore, correctly distinguishing patient response to cisplatin treatment would allow sensitive patients to benefit from chemotherapy and avoid non-responsive patients suffering from unnecessary side effects.
In our study, we for the first time identified that the expression of Notch1 and HES1 in osteosarcoma specimens were positively correlated with cisplatin sensitivity in osteosarcoma patients. In vitro study confirmed this finding that osteosarcoma cell line Saos-2 with higher Notch1 expression was more sensitive to cisplatin than MG63 with lower Notch1 expression. Subsequently, we consolidated such discovery by activating or suppressing Notch1 signaling pathway further. Activation of Notch1 signaling pathway significantly sensitized osteosarcoma cells to cisplatin treatment while inhibition of Notch1 signaling pathway desensitized these cells to cisplatin. We further revealed that changing the activity of Notch1 signaling pathway was able to affect the cell apoptosis to regulate the sensitivity of osteosarcoma to cisplatin, which might be mainly caused by the expression and/or activity changes of Caspase family proteins. As was shown in Figure 6, inhibition of Notch1 signaling pathway could significantly decrease the amount of Caspase family proteins, especially Pro-Caspase 3 and Cleaved-Caspase 3, after cisplatin treatment. Therefore, on the view of macroscopic, osteosarcoma cells showed decreased apoptosis and insensitivity to cisplatin. On the contrary, we speculated that activation of Notch1 signaling pathway would have the opposite effect on the expression and/or activity of Caspase family proteins, which could lead osteosarcoma more sensitive to cisplatin. Overall, our study comprehensively revealed that the expression of Notch1 was positively correlated with cisplatin sensitivity in osteosarcoma. Thus, Notch1 could be an effective molecular marker for predicting and even regulating the cisplatin sensitivity in osteosarcoma patients.
The highly conserved Notch signaling pathway plays important roles in controlling a wide variety of cell fate decisions and governs numerous developmental processes which affect the development and function of many organs. Notch signaling pathway has a consanguineous relationship with osteosarcoma in its pathogenesis, development, invasion, and metastasis in recent studies [23, 29, 30]. However, the influence of Notch signaling pathway on osteosarcoma chemotherapy sensitivity has hardly been reported before. Previous studies have reported activation of Notch1 signaling pathway was negatively related to cisplatin sensitivity in malignancies of head and neck squamous cell tumors [31, 32]. Opposite to these findings, we reported that activation of Notch signaling pathway was positively related with cisplatin sensitivity in osteosarcoma. Notch can have either an oncogenic [19–23] or a tumour-suppressor [33, 34] role. Thus, this might be epitomized by its different contributions to cancers in different tumor types. Therefore, we consider that the opposite findings from our study and previous studies may due to the different effects of Notch1 in different tumors. Osteosarcoma is a kind of malignancy derived from mesenchymal tissue. Therefore, we might also be able to comprehend the above results by drawing lessons from the effect of Notch signaling pathway in bone marrow mesenchymal stem cells (MSCs). Some researches showed that Notch signaling pathway undertook an important regulation action in the proliferation and differentiation of MSCs: activation of Notch signaling pathway could obviously inhibit the process of osteogenic and chondrogenic differentiation [35–38]. Thus, Notch signaling pathway would keep them in an undifferentiated or poorly differentiated state. Similarly, we would speculate that the activation of Notch1 signaling pathway may retain osteosarcoma cells in an undifferentiated stage, in which cell division and DNA replication process may be more frequent. When osteosarcoma cells are treated with chemotherapy drugs whose target is DNA, DNA damage and replication errors would be more likely to occur in this case, directly leading to the initiation of cell apoptosis. Indeed, poorly differentiated osteosarcoma cells tend to be more sensitive to chemotherapy in clinical. On the contrary, well-differentiated tumors are always insensitive to chemotherapy, which might be also the deeper reason for the phenomenon observed in our present study.
Cisplatin, one of the most common anti-neoplastic drugs, is always able to bring better prognosis for sensitive patients. Our study proved that Notch1 signaling pathway whose activity changes might directly or indirectly affect the expression and activity of Caspase family proteins was also a possible key regulator for cell apoptosis induced by cisplatin in osteosarcoma. That is to say, Notch1 signaling pathway possibly plays a vital role in the anti-neoplastic effect of drugs with potential apoptosis-inducing ability. However, the following points are still to be resovled about our research: 1) more patients samples should be collected to further verify the relationship between Notch1 signaling pathway and cisplatin sensitivity; 2) the intermediate molecular mechanisms on how Notch1 changing the expression and activity of Caspase family proteins should be better clarified; 3) more chemotherapy drugs with potential apoptosis-inducing ability should be involved to try to reveal the universal law.
Our findings showed that Notch1 signaling pathway had a positive correlation with cisplatin sensitivity in osteosarcoma. This novel finding adds to the understanding of osteosarcoma chemotherapy sensitivity. Notch1 signaling pathway can be used as a molecular marker for cisplatin sensitivity in osteosarcoma patients.
This work was supported by grant awarded from National Natural Science Foundation of China (No. 81071472 and No. 81371960), grant awarded by Biomedical Engineering crossover project of Shanghai JiaoTong University (YG2011ZD06), grant awarded for Innovative Research from Shanghai Municipal Education Commission (13YZ031) and grant for scientific research from the National Natural Science Foundation for the Youth of China (No. 81201364).
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