Bee venom induces apoptosis and suppresses matrix metaloprotease-2 expression in human glioblastoma cells

Abstract

Glioblastoma is the most common malignant brain tumor representing with poor prognosis, therapy resistance and high metastasis rate. Increased expression and activity of matrix metalloproteinase-2, a member of matrix metalloproteinase family proteins, has been reported in many cancers including glioblastoma. Inhibition of matrix metalloproteinase-2 expression has resulted in reduced aggression of glioblastoma tumors in several reports. In the present study, we evaluated effect of bee venom on expression and activity of matrix metalloproteinase-2 as well as potential toxicity and apoptogenic properties of bee venom on glioblastoma cells. Human A172 glioblastoma cells were treated with increasing concentrations of bee venom. Then, cell viability, apoptosis, matrix metalloproteinase-2 expression, and matrix metalloproteinase-2 activity were measured using MMT assay, propidium iodide staining, real time-PCR, and zymography, respectively. The IC₅₀ value of bee venom was 28.5 µg/ml in which it leads to decrease of cell viability and induction of apoptosis. Incubation with bee venom also decreased the expression of matrix metalloproteinase-2 in this cell line (p < 0.05). In zymography, there was a reverse correlation between bee venom concentration and total matrix metalloproteinase-2 activity. Induction of apoptosis as well as inhibition of matrix metalloproteinase-2 activity and expression can be suggested as molecular mechanisms involved in cytotoxic and antimetastatic effects of bee venom against glioblastoma cells.

Keywords:
Bee venom; Glioblastoma; Matrix metalloproteinase-2; Apoptosis; Metastasis; Zymography

Introduction

Glioblastoma is recognized as the most common malignant primary brain tumor with a particularly poor prognosis. Despite multiple therapeutic strategies such as surgery, radiotherapy, and chemotherapy, no effective treatment has been identified for glioblastoma (Haar et al., 2012; Naik et al., 2013). Moreover, resistance of brain tumors to available drugs has become a clinical challenge (Haar et al., 2012). Therefore, development of new natural therapeutic strategies is necessary. In different experiments, it was illustrated that the glioma cells show ability to produce and secrete various matrix metalloproteinases (MMP) enzymes (Rooprai and McCormick, 1996; Forsyth et al., 1999). It has been proposed that extracellular matrix degradation, triggered by MMP-2 activation via interaction with tissue inhibitor of metalloproteinase-2, is essential for invasion of glioma cells (Fillmore et al., 2001). Additionally, extracellular matrix degradation, especially by MMP-2, releases growth factors and provides more free spaces to vascular extension. Growth factors released by MMP such as vascular endothelial growth factor, fibroblast growth factor-2 and transforming growth factor beta may exert a significant effects in induction of angiogenesis (Egeblad and Werb, 2002). Therefore, protocols aiming to target MMP-2 activity may become a promising therapeutic strategy for treatment of glioma (Abe et al., 1994; Deryugina et al., 1997; Senota et al., 1998).

Bee venom (BV, apitoxin) is one of a natural biological complex compound with many different therapeutic effects including neuroprotective, anti-allergic, and anti-angiogenesis properties (Huh et al., 2010; Kim et al., 2011; Shin et al., 2014). The two main components of BV are melittin and phospholipase A₂. It has been reported that melittin has proapoptotic effect and shows anti-tumor activity (Oršolić, 2012). The BV has also been utilized in treatment of variety inflammatory conditions such as rheumatoid arthritis (Kwon et al., 2001; Park et al., 2004). These effects are shown to be mediated by inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells, mitogen-activated protein kinase and Ca²⁺/calmodulin signaling pathways (Cho et al., 2010; Park et al., 2010). Studies on glioblastoma cell lines revealed that disturbance of Ca²⁺/calmodulin signaling pathway could result in tumor cells apoptosis through inhibition of DNA synthesis (Tsuruo et al., 1982; Oršolić, 2009). Also, it has been previously reported that BV induces cell cycle arrest in human cervical cancer cells (Ip et al., 2008). In the present study, antiproliferative and apoptogenic properties of venom of honey bee on human A172 glioma cancer cells were investigated. Also, because of critical role of MMP (especially MMP-2) in invasion of glioblastoma (Lu et al., 2004), the possible inhibitory effects of BV on MMP-2 expression and activity were evaluated.

Material and methods

Materials

Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT), propidium iodide (PI), Triton X-100 and gelatin were purchased from Sigma (St. Louis, USA). RPMI-1640 media and fetal bovine serum (FBS) and penicillin-streptomycin solution were from Gibco (Life Technologies, Carlsbad, USA). The venom of honey bee (persica, worker bees) was purchased from Royan Zahr (Isfahan, Iran). Total RNA extraction kit, agarose gel, green viewer dye and the entire solvents and flasks were prepared from Parstous co (Iran). Human A172 glioblastoma and normal murine L929 fibroblast cell lines were obtained from Pasteur Institute, Iran.

Cell culture and treatment: The A172 and L929 cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and incubated in 37 °C and 5% CO₂. The BV stock solution (15 mM) was prepared in phosphate buffer saline, pH 7.2. For cell viability assay, the cells were seeded in 96-well culture plates (1 × 10⁴ cells/well). Then, the RPMI media was changed by fresh one containing varying concentrations (5–160 µg/ml) of BV or reference drug (cisplatin at 70 µg/ml). The cells were incubated for 24 or 48 h in 37 °C and 5% CO₂. For apoptosis assay, the cells were cultured in 6 well plates (1 × 10⁵ cells per well) and treated with BV at its IC₅₀ concentration for 48 h.

MMT assay: Effect of BV on A172 and L929 cell viability was determined using MTT assay as described previously (Mortazavian et al., 2012; Ghorbani et al., 2015). Briefly, 10 µl of MTT reagent (5 mg/ml) was added to each well, and the plates were incubated further for 4 h in 37 °C. At the end of incubation time, media was removed and formazan crystals were dissolved by adding 100 µl dimethyl sulfoxide. Finally, absorbance was read at 545 nm using ELISA plate reader (Stat fax-2100). The assay was carried out in triplicate.

Apoptosis analysis: After treatment with BV, the floating and adherent cells were harvested and incubated with a hypotonic buffer containing propidium iodide for 30 min (Mortazavian et al., 2013; Sadeghnia et al., 2014). The samples were then subjected to the flow cytometry for determination of apoptotic cells.

RNA extraction and cDNA synthesis: The cells were cultured in T25-flasks and treated with different concentrations of BV (0–10 µg/ml). Then total RNA was extracted using Parstous RNA extraction kit (Iran) according to the manufacturer's instruction. Quality of extracted RNA was checked by running on 1% agarose gel in the presence of cyber safe or green viewer (Parstous). Synthesis of cDNA was performed using Parstous kit according to the manufacturer's instruction.

Real time-PCR: MMP-2 primers were designed as follows: forward; 5′-AACTACGATGACGACAGCAAGT-3′ and reverse; 5′-AGGTGTAAATGGGTCCCATCA-3′. Quantitative RT-PCR was carried out on Stratagene 3000 instrument. Net volume of PCR reaction was 20 µl containing 1 µl cDNA, 1 µl mixed primer, 10 µl Sybergreen dye, 0.4 µl Rox dye and 7.6 µl distilled water. Temperature profile was designed as an initial denaturation phase at 95 °C (5 min). Following this, reaction continued in 35 cycles with temperature profile as denaturation (94 °C, 30 s), annealing (57 °C, 30 s) and extension (72 °C, 45 s). A final period of 72 °C for 5 min was considered to ensure maximum production of PCR products. The expression of MMP-2 gene was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene.

Gelatin zymography: To assess enzymatic activity of MMP-2, gelatin zymography was performed to identify 72 kDa (pro-MMP-2) and 62 kDa (active-MMP-2) isoforms of the protein. The cells were cultured in 96 well plates (1 × 10⁴ cells/well) for 12 h. Then they were washed with PBS, and subjected to treatment with different BV concentrations (0–10 µg/ml) in serum free medium for 24 h. To evaluate the direct effect of BV on MMP-2, different BV concentrations were added to the A172 cellular supernatant and incubated for 24 h. Then, the media were resolved on 8% SDS-PAGE containing 1% gelatin as enzyme substrate. The SDS was removed and the enzyme activity regenerated by washing the gels in 2.5% triton-X100 for three times. Subsequently, gels were incubated in developing buffer (Tris 50 mM pH 7.4, CaCl₂ 10 mM, NaN₃ 0.02% and sterile dH₂O) at 37 °C for 42 h. The area of digested gelatin was visualized by counterstaining using Coomassie brilliant blue R-250, and quantified as relative numerical values with arbitrary units using NIH ImageJ 1.42q software.

Statistical analysis

All results are presented as mean ± standard error of the mean (SEM). The values were compared using the one-way analysis of variance followed by Tukey's post hoc test for multiple comparisons. The p-values less than 0.05 were considered to be statistically significant.

Results

Effect of BV on cell viability

As shown in Fig. 1, BV decreased viability of glioblastoma cells in a concentration-dependent manner with the IC₅₀ value of 28.52 and 28.3 µg/ml for 24 and 48 h, respectively. After 24 h of incubation, viability of cells treated with 5, 10, 20, 40, 80 and 160 µg/ml of BV was 76 ± 3.5, 66 ± 2.5, 61 ± 0.5 (p < 0.05), 41 ± 0.5 (p < 0.001), 27 ± 0.5 (p < 0.001) and 25 ± 5% (p < 0.001) of control (100 ± 4%), respectively. At concentrations of 40–160 µg/ml, the antiproliferative effect of BV was more than that of 70 µg/ml cisplatin. This effect of BV was also more than cisplatin when the cells incubated for 48 h. Compared to untreated cells, cell viability was 70 ± 4.5, 63 ± 2.5, 60 ± 3 (p < 0.05), 41 ± 1 (p < 0.001), 25 ± 0.5 (p < 0.001) and 25 ± 1% (p < 0.001), respectively.

Fig. 1
Effect of bee venom on viability of human A172 glioblastoma cells. The cells were treated with different concentrations of bee venom or cisplatin (70 µg/ml) as reference drug for 24 or 48 h. *p < 0.05 versus control cells; ***p < 0.001 versus control cells.

The effect of BV on viability of L929 fibroblast cells is presented in Fig. 2. At the end of 48 h incubation, BV did not show any cytotoxic effect on normal fibroblast cells (Fig. 2).

Fig. 2
Effect of bee venom on viability of murine L929 fibroblast cells. The cells were treated with different concentrations of bee venom for 48 h.

Effect of BV on apoptosis

Effect of BV on apoptosis of glioblastoma cells is shown in Fig. 3. Flow cytometry analysis revealed that in control condition only 14 ± 5% of glioblastoma cells were in apoptosis stage. However, in the presence of 28.52 µg/ml (IC₅₀ value) of BV, percentage of the apoptotic cells was 64 ± 5% which is significantly higher than those in untreated cell population (p < 0.01).

Fig. 3
Effect of bee venom on apoptosis of human A172 glioblastoma cells. The cells were treated with bee venom for 48 h and then incubated with a hypotonic buffer containing propidium iodide and triton X-100 and analyzed with a flow cytometer. **p < 0.01 versus control cells (0 µg/ml).

Effect of BV on MMP-2 expression

RT-PCR analysis of MMP-2 expression showed that incubation with BV significantly reduced the level of MMP-2 mRNA in A172 cells. Compared to control untreated cells, level of MMP-2 mRNA fold changes were -0.1 and -2.1 at 0.1 and 1 µg/ml of BV concentrations, respectively (p < 0.05, Fig. 4).

Fig. 4
Effect of bee venom on MMP-2 gene expression. MMP-2 mRNA level was measured in untreated cells (control) and those exposed to 0.1 µg/ml and 1 µg/ml BV. *p < 0.05. GAPDH housekeeping gene served as internal control.

Effect of BV on MMP-2 activity

Treatment with BV significantly reduced the quantity of detectable MMP-2 enzyme in cellular supernatant (p < 0.001). Interestingly, ratio of active (62 kDa) isoform of MMP-2 was elevated by increasing BV concentration (Fig. 5).

Fig. 5
Effect of bee venom on the activity of MMP-2 enzyme in human A172 glioblastoma cells. (A) Evaluation of activities of pro-MMP-2 (72 kDa) and active MMP-2 (62 kDa) by gelatin zymography; (B) Quantitative presentation of MMP-2 activity (vertical axis represents gelatinolytic activity as arbitrary units. ***p < 0.001 versus control cells (0 µg/ml) regarding pro-MMP-2; ^## p < 0.01 versus control cells (0 µg/ml) regarding active MMP-2.

Discussion

It has been recently suggested that BV as a cytotoxic agent may has potential therapeutic effects in cancer (Ip et al., 2008; Oršolić, 2009, 2012). In present work, the effects of BV on cell viability, apoptosis and MMP-2 expression and activity were investigated on human A172 glioblastoma cells. Our data showed that BV treatment decreased cellular viability in a concentration-dependent manner through its proapoptotic action. This antiproliferative effect of BV at IC₅₀ value of 28.52 µg/ml was approximately comparable with the effect of cisplatin at 70 µg/ml. Furthermore, BV treatment did not affect viability of normal fibroblastic cells indicating a degree of specificity for malignant cells. Therefore, it seems that BV components can be good candidate for future clinical trials for cancer therapy. BV constitutes an enormous source of enzymes and bioactive peptides (e.g. melittin and phospholipase A₂), and its beneficial actions on tumor cells may be due the effects of a single constituent or by the effects of several of its constituents on the tumor cells (Oršolić, 2012).

Recently and in agreement with our findings, Gajski et al. reported that pre-incubation with BV induces cell sensitization to cisplatin, and therefore can improve the killing effect of this drug against human glioblastoma A1235 cells (Gajski et al., 2016). Also, Ip et al. demonstrated that BV induces cell cycle arrest and apoptosis in human cervical epidermoid carcinoma cells (Ip et al., 2008). The pro-apoptotic effect of BV can be mediated through intrinsic or extrinsic pathways, two general ways for activation of apoptosis. Intrinsic pathway is initiated by mitochondrial release of cytochrome c and subsequent activation of caspase-3. On the other hand, the extrinsic pathway is stimulated with a cell death receptor which activates caspase-8 and finally caspase-3. Activated caspase-3 targets substrates that promote DNA fragmentation (Kirkland et al., 2002; Ghorbani et al., 2015). It has been reported that proapoptotic effect of BV is mediated via a Fas receptor pathway involving mitochondrial dependent pathways which increased activation of caspase-3 and then lead to apoptosis (Ip et al., 2008).

The main characteristic feature of glioblastoma is local invasiveness. This feature makes it much difficult to resect tumor completely, and therefore patients have usually poor prognosis (Lu et al., 2004). This enhanced invasiveness is associated with increased production and secretion of MMPs enzymes (Rooprai and McCormick, 1996; Forsyth et al., 1999; Fillmore et al., 2001; Lu et al., 2004). Lu et al. (2004) showed an 80% increase in MMP-2 activation during invasion of human glioblastoma cells. The MMP-2 triggers extracellular matrix degradation, releases growth factors and provides more free spaces for vascular extension and cancer progression (Fillmore et al., 2001; Egeblad and Werb, 2002). Our data from Q-PCR and zymography showed an inverse relationship between BV concentration and MMP-2 expression and activity. Because the inhibitory effect of BV on MMP-2 activity was not seen in cell-free supernatant medium, this effect is most probably mediated by cellular signaling. It has been demonstrated that MMP-2 expression is under regulation of an intracellular signaling pathway known as extracellular-signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) (Stoica and Lungu, 2014). However, further studies are needed to reveal the exact mechanisms involved in altering MMP-2 expression and activity by BV.

The reduction of MMP-2 expression by BV suggests that this venom may have inhibitory effect on metastasis of tumors. This is in agreement with findings of Huh et al. (2010) who showed BV inhibits tumor angiogenesis and metastasis by inhibiting tyrosine phosphorylation of vascular endothelial growth factor in Lewis lung carcinoma-tumor-bearing mice.

In conclusion our results showed that BV inhibits viability of glioblastoma cells through induction of apoptosis. BV also decreased MMP-2 expression suggesting a potential role in inhibition of glioblastoma metastasis. Therefore, it can be good candidate for future clinical trials in glioblastoma tumors.

Authorship

Study conception and design: MS, BM, FZA, MM and MS. Acquisition of data: MS, AB and MZ. Analysis and interpretation of data: MS, HO, MN, HRS and AG. Drafting of manuscript: AB, FZA, BM, and Majid Mojarad. Critical revision: MS, HRS and AG.

Ethical disclosures

Protection of human and animal subjects

The authors declare that no experiments were performed on humans or animals for this study.

Confidentiality of data

The authors declare that no patient data appear in this article.

Right to privacy and informed consent

The authors declare that no patient data appear in this article.

Acknowledgments

This work was supported by a grant (911311) from Mashhad University of Medical Sciences (MUMS), and extracted from M.Sc. thesis presented by Mohsen Sisakht.

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Publication Dates

• Publication in this collection
  May-Jun 2017

History

• Received
  7 Aug 2016
• Accepted
  29 Nov 2016