Proteomic investigation of resistance to chemotherapy drugs in osteosarcoma

Abstract

BACKGROUND:

Osteosarcoma, which is also termed osteogenic sarcoma or osteoma sarcomatosum, is the most common form of bone cancer. Typical osteosarcoma can occur at any age, including in infants, children, and the elderly, but more than half of cases occur in individuals who are 10–20 years old.

OBJECTIVE:

Here, the objective was to search for protein markers to indicate resistance to cisplatin in osteosarcoma and provide a theoretical basis for the early and accurate use of cisplatin to treat osteosarcoma.

METHODS:

Thirty patients with osteosarcoma were selected for the study. Experimental studies on the chemosensitivity of osteosarcoma using an in vitro kit method were performed, and cisplatin-resistant and cisplatin-sensitive osteosarcoma tissues were obtained. A representative sample was chosen to analyze and identify differentially expressed proteins in cisplatin-resistant tissues.

RESULTS:

The osteosarcoma-sensitive tissue was analyzed using 2-D electrophoresis and time-of-flight mass spectrometry. Differently expressed proteins were analyzed by western blotting to identify markers. Cisplatin-resistant and cisplatin-sensitive osteosarcoma tissues were obtained. Five significantly differentially expressed proteins were identified, including ALDOA and PGK1.

CONCLUSIONS:

The results indicate that ALDOA and PGK1 might be appropriate markers that can be used when treating osteosarcoma with cisplatin.

Keywords

Osteosarcoma chemosensitivity proteomics cisplatin

1. Introduction

Osteosarcoma, which is also termed osteogenic sarcoma or osteoma sarcomatosum, is the most common form of bone cancer [1]. Typical osteosarcoma can occur at any age, including in infants, children, and the elderly; however, more than half of cases occur in individuals who are 10–20 years old, with a male to female ratio close to 2:1 [2]. Chemotherapy is one of the most important treatments for osteosarcoma [3]. Chemical medications help improve the success rate of limb salvage, often avoiding amputation. Chemotherapy is the best option for a good proportion of cases (about 66%) [4]; however, about 40% of patients, regardless of the medication used, experience treatment failure, and eventually, metastasis throughout the lungs will occur [5]. For patients undergoing chemotherapy, chemotherapy is no longer the initial therapy given. In cases of resistance to a particular chemotherapeutic drug, although the available drugs are different in their structure and function, they still tend to produce resistance to other chemotherapeutic drugs, ultimately resulting in widespread resistance. This phenomenon is called multidrug resistance (MDR) in medical science [6, 7].

There is an urgent need to develop a mechanism to ensure early diagnosis and guide drug use during chemotherapy for osteosarcoma in order to avoid wasting money and accurately time treatment. In the past 10 years, there has been much research on multidrug resistance in tumors; however, there has been little progress on MDR.

Cisplatin achieves its chemotherapeutic effect by inducing the activation of osteosarcoma cell autophagy. Therefore, it has become one of the three main drugs used for osteosarcoma neoadjuvant chemotherapy in the Rosen T10 protocol [8]. The present study not only involved a proteome analysis of bone cancer but also established resistance molecular markers for bone cancer. The clinical and pathological features of various types of bone cancer were considered in this study, and the specific expression patterns of proteins that can determine the various types of bone cancer were analyzed. The theoretical basis for the proper use of early chemotherapy drugs was established. The clinical applications of this technology may bring enormous social and economic benefits. The study on multi-drug resistance was performed as follows.

2. Samples

Thirty osteosarcoma tissue samples (from 19 males and 11 females, mean age, 18.5 years old) were taken at three hospitals: Zhuhai People’s Hospital, Zhuhai Second People’s Hospital, and the fifth affiliated hospital sun yat-sen university. Cisplatin, doxorubicin, paclitaxel, bleomycin, vincristine, and methotrexate were provided by the Oncology Department of the Zhuhai People’s Hospital. ATP chemotherapy drug sensitivity assay kits were purchased from Beijing Bauhinia Company.

3. Experimental methods

3.1 Analysis of osteosarcoma chemosensitivity using an in vitro kit method

3.1.1 Separation of osteosarcoma cells

The osteosarcoma tumor tissues were cut into fragments of roughly 1 mm $\times$ 1 mm $\times$ 1 mm with sterile ophthalmic scissors. At the same time, a digestive enzyme was mixed and dissolved in 10 mL CAM (Chorioallantoic Membrane) culture solution. Then, the solution was sterilized by filtration and 5–10 mL was placed in a sterile 50 mL centrifuge tube with a tissue sample. The tubes were incubated while tilted in a CO ${}_{2}$ incubator (37 ${}^{\circ}$ C, 3 h). About every 20 min, the tissue digestion fluids were mixed 5–10 times. While the tubes were shaken, the digestion progress of the cell suspension was observed by eye, and digestion was stopped when a clear cell suspension was observed. Then, the digestion mixture was gently pipetted repeatedly with a 10 mL tip to ensure that the tissue blocks had completely dispersed. After Trypan blue staining, the digestion status of the tumor cells was evaluated under a microscope.

3.1.2 Detection of chemosensitive osteosarcoma cells

The chemotherapeutic drug solutions were removed from storage in the refrigerator (200X concentrate), and they were prepared as follows. First, 500 $\mu$ L CAM broth was mixed with 20 $\mu$ L of each drug. Based on the number of cells obtained, six detectable concentrations were established, 200%, 100%, 50%, 25%, 12.5%, and 6.25% PPC, in the experimental wells while also establishing M0 drug-control wells. Then, 100 $\mu$ L CAM broth was added to the A-G line in each well using a multi-channel pipette. A variety of PPC test drugs were added using an adjustable pipette. To the M0 well, only 100 $\mu$ L CAM broth was added.

After Trypan blue staining, the total number of cells was determined. First, 2–3 cells/100 $\mu$ L were pipetted into each well, and before counting, the solution was mixed to avoid errors. Then, 100 $\mu$ L tumor cell suspension was added to the G-A line in each well using a multi-channel pipette. The 96-well plates were covered and fixed with tape and placed in a wet box and cultured for 5–7 d (37 ${}^{\circ}$ C, 5% CO ${}_{2}$ ). After 5–7 d of culture, a Bios stems fluorescence emission detector was used to measure ATP fluorescence, and drug sensitivity analysis was performed using drug-sensitive detection software.

The data are expressed as a percentage, and statistical analysis was performed using SPSS13.0 statistical software ( $P<$ 0.05 was considered statistically significant).

3.2 Detection of differentially expressed proteins using 2-D electrophoresis and time-of-flight mass spectrometry in vitro

3.2.1 The first phase of isoelectric focusing

(1) Clean water was used to rinse the adhesive strip slot, and the strip groove was carefully brushed to clean. The positive and negative electrodes were cleaned with a brush, and “Strip Holder Cleaning Solution” was used to clean the adhesive strip slots. Ultrapure water was then used to clean the slots, and they were allowed to air dry. (2) The hydration liquid was removed from storage at $-$ 20 ${}^{\circ}$ C, dissolved at room temperature, and prepared in water. (3) The sample volumes were calculated using the Brandford method. The volume of each protein solution in water was adjusted to 250 $\mu$ L. The samples were shaken, mixed, and then centrifuged for 30 min. (4) The plate electrode of the IPGphor was wiped with alcohol, which was allowed to evaporate completely before the plate electrode was used. (5) The rubber strips were freeze-dried at 20 ${}^{\circ}$ C in the refrigerator and equilibrated for 10 min at room temperature. (6) The rubber strip grooves were set parallel to the flat electrode of the IPGphor. The samples were placed evenly on the strip groove. The rubber strip was equilibrated at room temperature, and tweezers were used to gently remove the prefabricated dry film strip. The positive and negative strip poles were distinguished to ensure that the surface of the rubber strip faced downward in the strip groove. The rubber strip was imbibitioned for 15–30 min. (7) Then, 1 mL cover oil was added onto each strip. (8) The adhesive strip was placed on the slot, and the isoelectric focusing procedure was set. An appropriate amount of glue was selected. (9) After the rubber strip had focused, the second dimension SDS-PAGE electrophoresis step was immediately balanced and performed.

3.2.2 The second phase of SDS-PAGE electrophoresis

(1) The glass plate was repeatedly scrubbed with cotton dipped in detergent, rinsed with double distilled water, and allowed to air dry. (2) The glass plate was installed, and (3) two slabs of 12.5% acrylamide gel (85 mL) were made. (4) The solution was injected into the interlayer of the glass plate. (5) After solidifying, the gel was regularly replenished with deionized water. (6) Rubber buffer A was prepared. (7) An adhesive strip was placed on the mineral oil. (8) The gel was allowed to equilibrate for 13–15 min in equilibration buffer A. (9) The adhesive tape was removed, and the agarose gel was sealed. (10) The gel was allowed to sit for 15 min. (11) The gel was then transferred to the electrophoresis tank, and (12) electrophoresis was started. (13) Electrophoresis was stopped when the BPB indicator reached 0.5 cm from the bottom edge. (14) The two-dimensional gel was then placed in the storage solution.

3.2.3 Dyeing

(1) Silver staining kit contained the following: the fixing solution, sensitizing solution, staining solution, color liquid, and termination solution. (2) The following steps were used: Fixation, Sensibilization, Washing, Silver staining, Water flush, Termination, and Water flushing.

Analysis: (1) Gel scanning was carried out using a YLN-5000 electrophoresis scanning densitometer. (2) The results were analyzed using the software “Image Master 2D Platinum 7.0 Training”.

Protein mass spectrum identification: (1) The rubber was cut with the fully automatic “Exquest Spot Cutter (Biorad)” cutting system. (2) The adhesive proteins were selected, cut, and then collected together for rubber block analysis. The collected protein samples were sent to Shanghai Biological Company for protein mass spectrum identification.

3.3 Further in vitro detection of five differentially expressed proteins by western blotting

3.3.1 Tissue protein extraction

(1) The tissue blocks were weighed. (2) The tissue blocks were pulverized in liquid nitrogen using a mortar. (3) Then, 3 mL RIPA buffer and 30 $\mu$ L PMSF (10 mg/mL) were added to each gram of tissue. The mixtures were further homogenized using Polytron (15,000 rpm/min for 1 min) while maintaining the temperature at 4 ${}^{\circ}$ C. (4) PMSF was added (30 $\mu$ L/g tissue, 10 mg/ml PMSF), and the tissue samples were incubated on ice for 30 min. (5) They were then centrifuged at 4 ${}^{\circ}$ C for 15 min (about 15,000 rpm). (6) The supernatant of cell lysate was divided and stored at $-$ 20 ${}^{\circ}$ C. (7) Protein concentrations were determined using the Bradford colorimetric assay.

3.3.2 SDS-polyacrylamide gel electrophoresis

(1) Shelves were installed. (2) A separation gel was prepared (total: 8 ml), and a layer of distilled water was added to the surface of the gel to ensure good gel formation. (3) A concentrating gel was prepared after the separating gel had set (total: 3.5 ml). A pre-prepared comb was then inserted. (4) After the gel had set, the samples were added, and electrophoresis was started. A voltage of 60–80 V was used while the samples were in the upper gel, and the voltage was changed to 100–120 V when the samples moved into the separation gel. lectrophoresis typically took 1.5 h. Adding a layer of distilled water onto the surface of the gel to promote the glue agglutination.

3.3.3 Electrotransfer (semidrying process)

Selection of experimental conditions: A current density of 1 mA/cm ${}^{2}$ was used; 100 mA/film was usually used, and the transfer time was based on the size of the proteins and gel concentration. Appropriate adjustments were made based on the actual conditions.

Table 1
Number of patients resistant or sensitive to a variety of chemotherapy drugs following susceptibility testing

	Cisplatin	Doxorubicin	Paclitaxel	Bleomycin	Vincristine	Methotrexate
Resistance	8	7	8	6	6	20
Mildly sensitive	2	4	6	9	14	5
Moderately sensitive	15	16	10	12	8	5
Sensitive	5	3	6	3	2	0

Figure 1.

mages of human osteosarcoma cell growth after obtaining by separation. A: osteosarcoma cells (initial separation); B: osteosarcoma cells (cultured for 7 d), C: osteosarcoma cells (cultured for 14 d).

Experimental operation: (1) The filter paper and membrane were prepared. (2) Transfusion was blocked before the end of the transfer with prepared 5% milk (TBST solution). After blocking, the proteins were transferred to the film while in the milk solution. Clean filter paper was used for each transfer.

(1) Incubation with the primary antibody: The diluted antibody solution was incubated with the membrane, typically for 1 h; the incubation time was increased or decreased according to the volume and membrane antigen antibody. (2) The membrane was washed three times quickly with TBST to wash off the milk as soon as possible. The aim this wash step was to remove the primary antibody and non-specific antigen binding, and washing directly affected the results of the background staining. (3) The secondary antibody was incubated with the membrane for 1 h. Generally, HRP-conjugated secondary antibody was used diluted to 1:5000. Then, the membrane was washed three times quickly with TBST to wash off the milk as soon as possible (5 times, 5 min each). (4) The secondary antibody was exposed (HRP enzyme) to enhance the chemiluminescence.

4. Results

4.1 Chemosensitivity of osteosarcoma cells

4.1.1 Cultured of osteosarcoma tumor cells

The isolated osteosarcoma cells were circular in appearance, and they became fusiform after adhering. The number of cultured cells increased over time, as shown in Fig. 1.

4.1.2 Chemotherapeutic drug testing curves for osteosarcoma cells

Following chemotherapeutic drug testing, the inhibition curves of osteosarcoma cells showed that chemosensitivity differed among samples from different patients. The resistance rate and sensitivity rate decreased as the concentrations of the drugs decreased, as shown in Fig. 2.

Figure 2.

Chemotherapeutic drug testing curves for osteosarcoma cells. A: Six drug-resistant cases ; B: Some cases were drug sensitive (four were sensitive). Drugs 1 to 6 are cisplatin, doxorubicin, paclitaxel, bleomycin, vincristine, and methotrexate, respectively.

4.1.3 Testing patients for susceptibility to a variety of chemotherapy drugs

The numbers of cases of moderate and higher sensitivity for each group were as follows: Cisplatin group, 20 cases (66.7%); Doxorubicin group, 19 patients (63.3%); Paclitaxel group, 16 patients (53.3%); Bleomycin group, 15 cases (50%); Vincristine group, 10 cases (26.7%); and Methotrexate group, 5 cases (16.7%). The results for resistance were as follows: Cisplatin group, 8 patients (26.7%); Doxorubicin group, 7 patients (23.3%); Paclitaxel group, 8 patients (26.7%); Bleomycin group, 6 patients (20%); Vincristine group, 14 cases (46.7%); and Methotrexate group, 20 cases (66.7%). The rates of moderate or severe sensitivity in the Cisplatin and Adriamycin groups were significantly higher than those in the other groups. The sensitivity and resistance ratios for the Cisplatin and Paclitaxel groups were significantly higher than those for the other groups.

Table 2
Comparison of the results of the determination of protein expression gray values (gray value of detected proteins/gray value of internal reference protein, $\beta$ -actin) $p<$ 0.05, $n=$ 3

	PGK1	HMGB1	ENO1	ALDOA	GAPDH
Cisplatin-sensitive	0.78 $\pm$ 0.26	0.91 $\pm$ 0.02	0.48 $\pm$ 0.05	0.56 $\pm$ 0.12	2.56 $\pm$ 0.23
Cisplatin-resistant	1.36 $\pm$ 0.37	1.02 $\pm$ 0.023	0.52 $\pm$ 0.09	1.45 $\pm$ 0.15	2.79 $\pm$ 0.33

Figure 3.

Results of 2-D electrophoresis of cisplatin-sensitive and cisplatin-resistant osteosarcoma cells. A: Cisplatin-sensitive osteosarcoma cells; B: Cisplatin-insensitive osteosarcoma cells.

Figure 4.

Western blotting results for five related proteins in osteosarcoma cells. Lanes 1, 2, and 3: cisplatin-sensitive tissues; lanes 4, 5, and 6: cisplatin-resistant tissues.

4.2 2-D electrophoresis

After two-dimensional electrophoresis, scanning analysis was performed. A significant difference in the proteins between cisplatin-sensitive and cisplatin-insensitive osteosarcoma cells was found. The results are shown in Fig. 3. Five proteins that were significantly differentially expressed in osteosarcoma cells insensitive to cisplatin were identified by two-dimensional electrophoresis analysis. and analyzed by mass spectrometry. The five proteins were identified using a protein sequence database: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ENO1 (enolase 1), HMGB1 (high mobility group box 1), ALDOA (aldolase A), and PGK1 (phosphoglycerate kinase 1), The analysis results are shown in Fig. 3.

The expression of the five proteins described above was measured in three cisplatin-sensitive osteosarcoma cell lines and three cisplatin-resistant cell lines. The results showed that ALDOA and PGK1 were expressed significantly higher in cisplatin-resistant tissues than in cisplatin-sensitive tissues, and there was little difference in the expression of the three other proteins (Fig. 4 and Table 2).

5. Discussion

Currently, combined therapy including chemotherapy and surgery is the main approach to treat osteosarcoma. However, the emergence of multidrug resistance hinders the effectiveness of chemotherapy [9, 10, 11], and chemotherapy drug resistance in osteosarcoma is a serious problem [12, 13]. In this study, osteosarcoma cell strains resistant and sensitive to various chemotherapeutic drugs were established by the Kit method. As the primary drug studied in cancer research, the results for cisplatin in the susceptibility test were confirmed. A representative osteosarcoma cell strain that was resistant and sensitive to cisplatin was selected to perform 2-D pH gradient gel electrophoresis [14]. Total proteins were isolated from human osteosarcoma cells, and a proteome map was constructed. The differences between the cisplatin-resistant and cisplatin-sensitive cell strain on the proteome map were compared to determine the differentially expressed proteins. Using mass spectrometry, the corresponding peptide mass fingerprints were obtained [15]. A database was used to identify the proteins in the differentially expressed spots. Finally, the cells and pathological specimens from the patients were analyzed by western blotting to identify differentially expressed proteins [16]. ALDOA and PGK1 were identified as possible markers for cisplatin resistance when treating osteosarcoma.

6. Conclusions

Differences in the sensitivity to different chemotherapy drugs in osteosarcoma were identified. The expression levels of five proteins were found to differ between cisplatin-resistant and cisplatin-sensitive osteosarcoma tissues. It was concluded that ALDOA and PGK1 may be the appropriate markers of cisplatin resistance when treating osteosarcoma.

Footnotes

Acknowledgments

This work presented in this paper is supported by The Sichuan Province Department of Science and Technology under Grant 2015JY0119, the Key Fund Project of Sichuan Provincial Department of Education under Grants (17CZ0005, 17ZA0047). The Engineering and Technical College of Chengdu University of Technology under Grants (C122015005, C122016030). The Leading Talent Training Project of Neijiang Normal University (2016 [Liu Yi-He]).

Conflict of interest

The authors confirm that this article content has no conflict of interest.

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