NF-κB plays a pivotal role in tumourigenesis and tumour progression
[5, 6]. Aberrant or constitutively activated NF-κB has been detected in human glioblastomas
[15, 16]. The sesquiterpene lactone parthenolide inhibits NF-κB by preventing the degradation of IκB-α and IκB-β
. We investigated the anti-invasive and anti-angiogenic effects of parthenolide on glioblastoma cells for the first time by using two PTEN-mutant glioblastoma cell lines.
Glioblastoma invasion into normal brain tissues involves disruption of the extracellular matrix (ECM) and the subsequent penetration of tumour cells into adjacent brain structures. This process is partly mediated by the tumour secretion of MMPs, a group of enzymes necessary for ECM degradation and involved in reconstructing ECM components and accelerating tumour cell migration
. Through their function as proteolytic enzymes, MMPs destroy the collagen components of the basement membrane (BM) and the ECM, thus disrupting intracellular adhesion, enabling tumour cell invasion, and promoting tumour progression. Elevated levels of MMPs, especially MMP-2 and MMP-9, correlate with glioblastoma tumour aggressiveness, and are believed to play an important role in tumour cell invasion
[18–21]. In vivo and in vitro studies have revealed that the inhibiting MMP expression significantly reduces the invasive capacity of glioblastoma cells
. Dimerisation of the NF-κB transcription factor at the κB sequence in the MMP-9 promoter initiates MMP-9 transcription, thus upregulating MMP-9 protein expression
. Therefore, blocking NF-κB transcriptional activity by parthenolide may inhibit glioblastoma cell invasion. We found that parthenolide treatment inhibits both glioblastoma cell invasion and MMP-9 expression. These data suggest that parthenolide inhibition of glioblastoma cell invasion is mediated by NF-κB inhibition.
Tumour-induced angiogenesis is critical for the growth of solid tumours. Numerous studies have reported a correlation between increased intratumour microvessel density (MVD) and the risk of metastasis and/or decreased survival of patients with solid tumours
[24–26]. NF-κB is an important upstream regulator of VEGF
, a major angiogenic factor that induces endothelial cell proliferation. Glioblastomas are characterised by a high level of angiogenesis, and both VEGF secretion from tumour cells and TNF-alpha-induced NF-κB activation in endothelial cells promote endothelial cell survival by increasing anti-apoptotic gene expression under conditions of serum starvation. These pathways may, therefore, contribute to the maintenance of glioblastoma angiogenesis
. Aberrant MMP-9 expression is also implicated in the glioblastoma angiogenesis process. Low basal MMP-9 expression occurs in normal brain tissue, but high expression levels are induced in glioblastomas, and are linked to increased tumour cell proliferation
. We found that parthenolide suppresses both tumour cell-induced angiogenesis and expression of the NF-κB targets, i.e., VEGF and MMP-9, in glioblastoma cells. These data suggest that parthenolide suppresses tumour-induced angiogenesis through NF-κB inhibition.
With regard to tumor proliferation, we observed that parthenolide inhibits glioblastoma cell proliferation in dose-dependent manner. In the present study, cell viability (MTT assay/48h) of U87MG cells decreased to 85 % and 45 %, after the addition of 5 and 10 μM parthenolide, respectively, compared to the control. Zanotto-Filho et al. also reported that parthenolide inhibits glioblastoma cell proliferation in dose-dependent fashion and that NF-κB inhibition by parthenolide significantly correlated in decreases in cell viability of glioblastoma cells
. In their report, cell viability (MTT assay/36h) of U138MG cells decreased to 88 % and 34%, after the addition of 5 and 25 μM parthenolide, respectively, compared to the control. Our results were close to their research data. On the contrary, Anderson et al. performed proliferation assay using not the MTT but the CCK8 reagent. They reported that cell viability (CCK8 assay/24h) of U87MG cells decreased to about 78% and about 68%, after the addition of 5 and 10 μM parthenolide, respectively, compared to the control and that the inhibitory effect reached the plateau even if the concentration of parthenolide was raised exceeding 20 μM
. And they also reported that parthenolide was only able to suppress NF-κB activity by 20-30%. The reason why the inhibitory effect reached the plateau was not clarified. On the other hand, we agree with their opinion that another pathway other than NF-κB is also involved in antitumor effect of parthenolide.
By the way, NF-κB mediates the expression of anti-apoptotic and pro-apoptotic proteins. And most studies have attributed parthenolide suppression of tumour cell proliferation to the induction of apoptotic proteins mediated by NF-κB inhibition. In colorectal cancer cells, for example, conformational changes in Bax and upregulation of Bak lead to mitochondrial dysfunction and the induction of apoptosis
. Recent reports suggest that multiple pathways are involved in parthenolide-induced apoptosis in human cancer cells, including oxidative stress, intracellular thiol depletion, endoplasmic reticulum stress, caspase activation, and mitochondrial dysfunction
[31–33]. In addition, parthenolide inhibition of c-Jun N-terminal kinase (JNK) sensitises JB6 murine epidermal cells to ultraviolet B (UVB)-induced apoptosis, suggesting an anti-apoptotic role for JNK
. Another report suggests that parthenolide- induced transcriptional suppression of pro-apoptotic genes is mediated by STAT inhibition and acts at both the transcriptional level and by direct inhibition of IKK-β
In the present study, we used U87MG and U373 glioblastoma cells containing constitutively activated NF-κB due to PTEN mutation to examine cell survival and apoptosis. We demonstrated that parthenolide inhibits Akt phosphorylation, upregulates Bak and Bax, and activates caspase-3 and caspase-9. These results suggest that parthenolide induces apoptosis in glioblastoma cells by activating the mitochondrial apoptosis cascade and reducing survival signals through the inhibition of the Akt pathway. Parthenolide was previously reported to induce apoptosis in colorectal cancer and cholangiocarcinoma cells via the mitochondrial pathway
[30, 36]. In contrast, NF-κB inhibition by parthenolide markedly enhances the sensitivity of resistant breast cancer tumour cells to tamoxifen through suppression of the Akt pathway
. Taken together, the pro-apoptotic effect of parthenolide includes both stimulating the intrinsic apoptotic pathway and modulating expression of Bcl-2 family proteins.
In our in vivo study, parthenolide inhibited the growth of transplanted glioblastoma cells in mouse xenografts. This suggests that inhibitory effect of parthenolide on PTEN-mutant glioblastoma cells is caused by a combination of three mechanisms: suppression of tumour cell invasion, suppression of angiogenesis, and induction of tumour cell apoptosis.
We investigated tumour-induced angiogenesis by using a new in vitro angiogenesis assay that measures EC tube formation in collagen gels. Tumour-induced angiogenesis is usually examined by adding tumour cell medium directly to ECs without direct contact between the endothelial and tumour cells. In contrast, the new method allows the exchange of secreted factors between the endothelial and tumour cells. HUVECs have been used for many angiogenesis studies, especially those including in vitro experiments. However, microvascular endothelial cells from the organ being studied are presumed to be the most appropriate tool for tumour angiogenesis assays. Therefore, for the first time, we investigated the effect of parthenolide on angiogenesis by using HBMECs.
The effect of parthenolide treatment on neuro-inflammatory disorders has been examined in the recent years
[38–40]. Runmel et al. investigated the potential of parthenolide to reduce brain inflammation and reported that parthenolide can cross the blood–brain barrier
. Thus, the combined antitumour and anti-inflammatory properties of parthenolide make it a promising candidate for further studies of neurological diseases.