Procaine Inhibits Proliferation and Migration and Promotes Cell Apoptosis in Osteosarcoma Cells by Upregulation of MicroRNA-133b.

Procaine (PCA) is a conventional chemotherapeutic agent for osteosarcoma. Recent studies have proposed that the growth-inhibitory effect of PCA is through regulation of microRNAs (miRNAs). miR-133b has been proven to be a tumor suppressor in osteosarcoma, but whether it is involved in the antitumor effects of PCA on osteosarcoma has not been investigated. In this study, we aimed to explore the effects of PCA on osteosarcoma MG63 cells by regulation of miR-133b, as well as its underlying mechanisms. MG63 cells were treated with different concentrations of PCA, and cell viability, apoptosis, and miR-133b expression were then detected by MTT, flow cytometry, and qRT-PCR, respectively. Cells were then transfected with the miR-133b inhibitor and treated with 2 μM PCA. Thereafter, cell viability, migration, and apoptosis were detected. Analysis of signaling pathways was detected by Western blot. Our results showed that PCA significantly inhibited cell viability and promoted apoptosis and the expression level of miR-133b in a dose-dependent manner (p < 0.05 or p < 0.01). Moreover, we observed that PCA + miR-133b inhibitor dramatically reversed the effects of PCA on cell viability, apoptosis, and migration (p < 0.05 or p < 0.01). In addition, PCA significantly decreased the levels of p/t-AKT (p308 or p473), p/t-ERK, and p/t-S6, whereas PCA + miR-133b inhibitor rescued these effects. Our results suggest that PCA inhibits proliferation and migration but promotes apoptosis in osteosarcoma cells by upregulation of miR-133b. These effects may be achieved by inactivation of the AKT/ERK pathways.

INTRODUCTION

Osteosarcoma is the most prevalent primary malignant bone tumor, which arises from osteoid tissue, mainly affecting children and adolescents1. Although osteosarcoma is relatively rare, it has been estimated that it is the third most frequent malignancy in adolescence, followed by lymphomas and brain tumors in this age group1. The disease is generally locally aggressive, and patients often tend to develop early systemic metastases1,2. Although dramatic improvements have been made in the treatment of osteosarcoma in the past two decades, the survival rate still remains poor. It has been reported that the 5-year survival rate of osteosarcoma is about 20%3 because recurrence often manifests as metastasis4–6. Therefore, there is an urgent need to explore a novel strategy that would effectively inhibit tumor cell growth and metastasis.

Recently, accumulating evidence has focused on the administration of a combination of chemotherapeutic drugs to treat malignancy7–9. Procaine (PCA), one of the conventional chemotherapeutic agents, was also one of the most widely used local anesthetics during surgery. PCA has recently been proven to be a DNA-demethylating agent, which could demethylate densely hypermethylated CpG islands10. Thus, PCA has become one of the most promising choices for the treatment of different types of cancers, such as lung cancer11,12, liver cancer13, colon cancer14, and leukemia15. However, little information is currently available on the effects of PCA with different doses on osteosarcoma cells.

MicroRNAs (miRNAs) are a class of highly conserved, endogenous, small (∼22 nt), noncoding RNAs16. An increasing number of studies have confirmed that miRNAs are involved in cancer development and progress, which could regulate cell proliferation, apoptosis, differentiation, and metastasis17. The functional role of miRNAs in osteosarcoma has been widely studied18,19. Among the miRNAs, miR-133b was proven to be expressed in a lower level in osteosarcoma samples, which might be by regulating cell proliferation and cell cycle20. Therefore, we assumed that there might be a potential association between PCA and miR-133b.

Thus, in the present study, we aimed to explore the effects of PCA on osteosarcoma cell proliferation, apoptosis, and migration, as well as the relationship between PCA and miR-133b. In addition, we investigated the potential underlying signaling pathway(s). Our study might provide a new insight into the treatment of osteosarcoma.

MATERIALS AND METHODS

Cell Culture and Treatment

The human osteosarcoma cell line MG63 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were maintained in RPMI-1640 (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies), 1% penicillin (50 U/ml)/streptomycin (50 μg/ml) (Gibco Life Technologies), and 5 μg/ml Plasmocin™ prophylactic (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a humidified atmosphere of 5% CO2. The cells were cultured for 24 h, followed by administration of PCA (Sigma-Aldrich) at different concentrations (0.5, 1, 1.5, and 2 μM). Nontreated cells were considered to be the control group. The culture medium was changed every 24 h.

Cell Transfection

Mature miR-133b inhibitor and negative control were designed and synthesized by GenePharma (Shanghai, P.R. China). For stable transfection, cells were plated onto six-well plates at 2 × 105 cells/well and were then transiently transfected with 150 nM miR-133b inhibitor or negative control. Cell transfections were performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.

Cell Viability

Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2 5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay, according to standard methods described previously21. Briefly, the cells were seeded into 96-well plates and then exposed to PCA and/or transfected with miR-133b inhibitor or negative control. Seventy-two hours later, 5 mg/ml MTT (20 μl; Sigma-Aldrich) was added, and the cells were incubated at 37°C for another 4 h. Next, 100 μl of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to the cells to dissolve the formazan crystals. Thereafter, an absorbance at 590 nm was read by a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Hercules, CA, USA). Each experiment was performed three times.

Apoptosis Assay

The cell apoptosis was measured by annexin V-FITC/PI double staining using the FITC Annexin-V Apoptosis Detection Kit (Beijing Biosea Biotechnology, Beijing, P.R. China). In brief, cells were seeded into six-well plates at a density of 1 × 105 cells/well. The cells were treated with PCA and/or transfected with miR-133b inhibitor or negative control. Thereafter, the cells were washed twice with cold phosphate-buffered saline (PBS), followed by adding a solution containing 5 μl of annexin V-FITC plus 10 μl of PI. After incubation in the dark for 15 min, 300 μl of binding buffer was added to the cells. The cells were then analyzed by a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA).

Migration Assay

Cell migration was determined using a Transwell chamber with a pore size of 8 mm. In brief, MG63 cells were suspended in serum-free medium (200 μl) and then seeded on the upper compartment of a 24-well Transwell culture chamber. Thereafter, complete medium (600 μl) was added to the lower compartment and incubated for 12 h. The cells were then fixed with methanol, and nontraversed cells were removed from the upper surface with a cotton swab. Cells migrating to the lower side of the filter were stained with crystal violet and then counted.

Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR)

Total RNA was isolated from the cells using TRIzol reagent (Invitrogen). Reverse transcription was performed using the PrimeScript™ RT Master Mix (Takara, Otsu, Japan). The PCR primers were designed by GenePharma. PCR was conducted by Bio-Rad CFX 96 Real-time PCR system (Bio-Rad, Hercules, CA, USA) using SYBR® Premix Ex Taq™ II (TaKaRa, Shiga, Japan), according to the manufacturer’s instructions. miRNA and mRNA levels were normalized to U6 and GAPDH, respectively.

Western Blot

After treatment with PCA or transfection with miR-133b inhibitor and negative control, cell suspension was collected, centrifuged, and lysed in a RIPA lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) with protease and phosphatase inhibitor cocktails (Roche, Guangzhou, P.R. China). The protein concentration was quantified using the Bradford assay (Thermo Fisher Scientific). Equal amounts of protein were subjected to 10%–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and then transferred onto polyvinylidene fluoride (PVDF) membranes. The transfer membranes were then incubated with 5% skim milk in Tris-buffered saline with Tween (TBST) for 2 h and probed with the following primary antibodies overnight at 4°C at a dilution of 1:1,000: anti-Bax antibody (ab32503; Abcam, Cambridge, MA, USA), anti-B-cell lymphoma (Bcl)-2 antibody (ab32124; Abcam), anti-procaspase 3 antibody (ab32150; Abcam), anti-cleaved caspase 3 antibody (ab13585; Abcam), anti-phosphor (p)-AKT (Thr308) (ab38449; Abcam), anti-p-AKT (Ser473) (P4112; Sigma-Aldrich), anti-AKT antibody (#4691; Cell signaling Technology, Danvers, MA, USA), anti-extracellular signal-regulated kinase (ERK) antibody (ab214362; Abcam), anti-total ERK antibody (ab196883; Abcam), anti-p-S6 antibody (ab186753; Abcam), or anti-S6 antibody (SAB5500173; Sigma-Aldrich). Thereafter, membranes were incubated with a secondary antibody marked by horseradish peroxidase for 2 h at room temperature. The signals were captured with the WEST-ZOL (plus) Western Blot Detection System (Intron Biotechnology, Inc., South Korea) using enhanced chemiluminescence (ECL) reagents.

Statistical Analysis

All experiments were repeated at least three times. The data are presented as the mean ± standard deviation (SD). SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA statistical software) was used to conduct statistical analyses. One-way analysis of variance (ANOVA) was performed to calculate the p values. A value of p < 0.05 was considered as a statistically significant result.

RESULTS

PCA Inhibits Cell Viability and Promotes Cell Apoptosis in MG63 Cells

To investigate the function of PCA in osteosarcoma, the human osteosarcoma cell line MG63 was exposed to different concentrations of PCA (0.5, 1, 1.5, and 2 μM). Nontreated cells were regarded as the control group. Thereafter, cell viability and cell apoptosis were explored. Results showed that cell viability was decreased by the administration of PCA, but there were significant differences at 1.5 and 2 μM (both p < 0.05) (Fig. 1A). In addition, the percentage of apoptotic cells was significantly increased by 1, 1.5, and 2 μM PCA (all p < 0.05). Although the apoptotic cells were increased by 0.5 μM PCA, there was no significant difference (Fig. 1B). The results demonstrated that PCA inhibited cell viability and promoted cell apoptosis in MG63 cells in a dose-dependent way.

Figure 1

Procaine (PCA) inhibits cell viability and promotes cell apoptosis in MG63 cells. (A) Cell viability was significantly decreased by PCA at 1.5 and 2 μM. (B) Cell apoptosis was significantly increased by PCA at 1, 1.5, and 2 μM. *p < 0.05, **p < 0.01.

PCA Promotes Expression of miR-133b

Some studies have demonstrated that the effects of PCA might be by regulation of miRNAs22. However, whether miR-133b is involved in the antitumor effects of PCA on osteosarcoma has not been investigated. To investigate the functional role of miR-133b in PCA activity, the expression levels of miR-133b were analyzed after exposure to PCA. Results revealed that the expression levels of miR-133b were significantly elevated by PCA at 1.5 μM (p < 0.05) and 2 μM (p < 0.01) (Fig. 2). However, no significant differences were observed at 0.5 and 1 μM. The results implied that PCA promoted the expression levels of miR-133b in a dose-dependent way. Therefore, 2 μM was used in future studies.

Figure 2

PCA promotes the expression of miR-133b. The results revealed that the expression levels of miR-133b were significantly elevated by PCA at 1.5 and 2 μM. miR, microRNA. *p < 0.05, **p < 0.01.

PCA Inhibits Cell Viability by Regulation of miR-133b

To further investigate the function of miR-133b in osteosarcoma, miR-133b inhibitor was transfected into MG63 cells to suppress miR-133b expression. Results displayed that the levels of miR-133b was markedly reduced by miR-133b inhibitor (p < 0.01), indicating that the transfection efficiency was high and could be used for further study (Fig. 3A). Thereafter, cell viability was again analyzed by MTT. Results demonstrated that cell viability was statistically reduced by PCA, as expected, compared to the control group (p < 0.05). However, the effects were reversed by simultaneous transfection with miR-133b inhibitor (Fig. 3B). Cell viability was significantly increased by PCA + miR-133b inhibitor compared to the PCA + inhibitor NC group (p < 0.05). These results implied that PCA inhibited cell viability by regulation of miR-133b.

Figure 3

PCA inhibits cell viability by regulation of miR-133b. (A) The levels of miR-133b were markedly reduced by the miR-133b inhibitor. (B) Cell viability was statistically increased by PCA + miR-133b inhibitor compared to the PCA + inhibitor NC group. NC, negative control. *p or #p < 0.05, **p < 0.01.

PCA Promotes Cell Apoptosis by Regulation of miR-133b

We then analyzed whether PCA promotes cell apoptosis by regulation of miR-133b. We observed that cell apoptosis was statistically elevated by PCA, as expected, compared to the control group (p < 0.01). However, the effects were rescued by simultaneous transfection with the miR-133b inhibitor. PCA + miR-133b inhibitor significantly decreased cell apoptosis compared to the PCA + inhibitor NC group (p < 0.05) (Fig. 4A). We further explored the underlying mechanism of apoptosis by measuring the mRNA and protein expression levels of apoptosis-related proteins (Bax, Bcl-2, pro-caspase 3, and cleaved caspase 3). We observed that PCA significantly increased the mRNA expression of Bax and cleaved caspase 3 but decreased the level of Bcl-2 (p < 0.05 or p < 0.01). However, these effects were reversed by transfection with the miR-133b inhibitor (p < 0.05 or p < 0.01) (Fig. 4B). Protein levels were in line with the mRNA expression (Fig. 4C). These results indicated that PCA promoted cell apoptosis by regulation of miR-133b.

Figure 4

PCA promotes cell apoptosis by regulation of miR-133b. (A) PCA + miR-133b inhibitor significantly decreased the cell apoptosis compared to the PCA + inhibitor NC group. (B, C) The mRNA and protein expression levels of apoptosis-related protein (Bax, Bcl-2, procaspase 3, and cleaved caspase 3). Bcl-2, B-cell lymphoma-2. *p or #p < 0.05, **p or ##p < 0.01.

PCA Inhibits Cell Migration by Regulation of miR-133b

We next analyzed whether PCA inhibited cell migration by regulation of miR-133b. Results revealed that cell migration was statistically lowered by administration of PCA compared to the control group (p < 0.01) (Fig. 5). However, the effects were reversed by simultaneous transfection with the miR-133b inhibitor. These results implied that PCA inhibited cell migration by regulation of miR-133b.

Figure 5

PCA inhibits cell migration by regulation of miR-133b. The cell migration was statistically elevated by PCA + miR-133b inhibitor compared to the PCA + inhibitor NC group. **p or ##p < 0.01.

PCA Inactivates the AKT/ERK Pathways by Regulation of miR-133b

AKT/ERK pathways have been reported to be involved in cell proliferation, apoptosis, and migration23. Therefore, we assumed that AKT/ERK pathways might be responsible for the effects of PCA on MG63 cells. To confirm this assumption, we measured both the mRNA and protein expression of AKT/ERK pathway key factors. Results displayed that the mRNA levels of p/t-AKT (p-308 or p473), p/t-ERK, and p/t-S6 were significantly decreased by PCA compared to the control group (all p < 0.05). In addition, PCA + miR-133b inhibitor significantly elevated the above levels compared to the PCA group (all p < 0.05) (Fig. 6A). The protein levels showed results similar to the mRNA levels (Fig. 6B).

Figure 6

PCA inactivates AKT/ERK pathways by regulation of miR-133b. (A) mRNA and (B) protein levels of p/t-AKT (p308 or p473), p/t-ERK, and p/t-S6 were significantly decreased by PCA, whereas PCA + miR-133b inhibitor significantly elevated these levels. *p or #p < 0.05, **p < 0.01.

DISCUSSION

In the present study, we observed that PCA inhibited cell viability and promoted cell apoptosis in MG63 cells in a dose-dependent way. Moreover, the results revealed that PCA could elevate the expression of miR-133b. Furthermore, our study suggested that the effects of PCA on cell viability, apoptosis, and migration were by upregulation of miR-133b. In addition, the results demonstrated that PCA inactivated the AKT/ERK pathways, which were also by upregulation of miR-133b.

Currently, extensive chemotherapy and surgical resection are main strategies in the treatment of osteosarcoma. PCA as a fast and efficient local anesthetic and conventional chemotherapeutic agent has become popular in the treatment of different types of cancers10. Recent studies have reported that PCA could influence cancer cell proliferation, DNA methylation, and tumor progression12,13,24. In consideration of abnormal cell proliferation, higher metastasis, and DNA methylation in osteosarcoma25–27, we assumed that PCA may protect against osteosarcoma. Hence, in the present study we focused on the effects of PCA on the treatment of osteosarcoma, along with the underlying mechanisms. Consistent with previous studies12–14, our study confirmed that PCA could inhibit cell viability and stimulate cell apoptosis in a dose-dependent way, indicating a protective effect from PCA in osteosarcoma.

To further investigate the underlying mechanism by which PCA induced cell growth arrest and promotion of cell apoptosis, we examined whether PCA could affect miR-133b expression because miR-133b has been regarded to be a tumor suppressor in a variety of cancer cell lines such as esophageal squamous cell carcinoma (ESCC)28, colorectal cancer29, gastric cancer30,31, and osteosarcoma32. Previous studies confirmed that miR-133b potently inhibited cell proliferation, migration, and invasion and promoted apoptosis in osteosarcoma20,32. Based on previous studies, we hypothesized that the effects of PCA on MG63 cells might be by upregulation of miR-133b. As expected, the results revealed that the expression of miR-133b was significantly increased by PCA in a dose-dependent way. Furthermore, we analyzed the effects of the combination of PCA and suppression of miR-133b on cell viability and apoptosis again, together with migration. Our data showed that the combination of PCA and suppression of miR-133b significantly reversed the effects of PCA on cell viability, apoptosis, and migration, suggesting that the effects of PCA on osteosarcoma cells were achieved by upregulation of miR-133b.

To further understand the underlying mechanism of the apoptosis-promoting role of miR-133b in osteosarcoma cells, we investigated the expression of apoptosis-related protein. The Bcl-2 family plays a significant role in mediating cell apoptosis and consists of antiapoptotic and proapoptotic members33. Bcl-2, an important antiapoptotic factor, is highly expressed in many kinds of cancers and contributes to tumor initiation, progression, and resistance to treatment, while Bax is a proapoptotic protein34. In addition, caspases are crucial regulators of cell apoptosis35. Activation of caspases needs cleavage of procaspases. For example, procaspase 3 is activated by cleavage into cleaved caspase 3, the executioner of apoptosis36. In the present study, we observed that PCA markedly increased the level of Bax and cleaved caspase 3, whereas it decreased Bcl-2, implying an apoptosis-promoting role for PCA. However, the result was reversed by simultaneous suppression of miR-133b. Our findings are in line with the earlier observation of Patron et al., who suggested that miR-133b targeted antiapoptotic genes and increased apoptosis37.

In addition, we tested the potential signaling pathway(s). AKT and ERK signaling pathways are important survival pathways for cell proliferation, apoptosis, and migration in cancer cells38,39. Inhibition of the AKT and ERK signaling pathways could inhibit cell growth, induce apoptosis, and suppress migration40,41. Herein we found that PCA decreased the expression of p-AKT and p-ERK, suggesting that AKT and ERK inactivation might be required in PCA-induced cell proliferation apoptosis and migration. However, these effects were alleviated by simultaneous suppression of miR-133b. In other words, suppression of miR-133b might activate AKT pathways, while overexpression of miR-133b may inactivate the AKT pathway. Similarly, Zhao et al. suggested that p-AKT was reduced in miR-133b-overexpressed cells32. Additionally, the data showed that PCA decreased the level of p-S6, while simultaneous suppression of miR-133b raised the levels of p-S6. p-S6 is a downstream target of p70S6K, which has been implicated as an oncogene. These results imply that PCA also protected against osteosarcoma by regulating the expression of p70S6K. However, further studies should be performed to confirm the results.

In conclusion, our study suggests that PCA prevents proliferation and migration and enhances cell apoptosis in osteosarcoma cells by upregulation of miR-133b, which might be by inactivation of the AKT/ERK pathways. Our study provides basic theories for PCA in the treatment of osteosarcoma.