Mifepristone improves chemo-radiation response in glioblastoma xenografts

Glioblastoma multiforme (GBM) is the most common tumor of the central nervous system, with a prognosis of 15 months median survival following diagnosis. GBM is a fast-growing glioma that develops from astrocytes, star-shaped glial cells that support nerve cells. GBM is classified as a grade IV astrocytoma, which is the most invasive type of glial tumor. This kind of tumor is highly aggressive, growing rapidly and commonly spreading to nearby brain tissue. Its treatment has been a challenge due to its localization in the brain [1, 2].

The standard treatment for GBM is surgery, followed by radiation therapy [3, 4] accompanied by chemotherapy. In standard external radiation therapy (radiotherapy), multiple sessions of standard-dose "radiation fractions" are delivered to the tumor site as well as its margin in order to treat the zone of infiltrating tumor cells. Although the goal of radiation therapy is to kill tumor cells selectively while leaving normal brain tissue unharmed, in reality each treatment session induces damage to both healthy and normal tissue, which limits the benefit of radiotherapy.

The radiation dose that can be tolerated by the brain is approximately 60 Gray. However, this dose is inadequate for total tumor eradication, resulting in a poor treatment response in patients with glioma. Consequently, the co-administration of chemotherapy with radiation has been used in recent years with the intention of improving treatment response [5, 6].

Chemotherapy with the drug temozolamide is the current standard treatment for GBM. The drug is administered every day during radiation therapy and then in six to eight cycles of five days (with a rest period between each cycle) at higher doses once radiation is completed [7]. While the aim of chemotherapy is long-term tumor control, this goal is reached in only about 20 percent of patients. That is overall patient survival with prolonged remission of GBM tumors has not improved with the addition of chemotherapy to the treatment regimen [8, 9]. Frequency of recurrence and rapid tumor progression emphasize the need for treatment alternatives to achieve long-term patient survival. Research efforts currently underway are focused on two approaches: (i) inhibition of angiogenesis in tumors, and (ii) the identification of agents that effectively and preferentially trigger the cell death process in tumors.

The activation of angiogenesis, the formation of new blood vessels from a preexisting vascular network, appears to play an important role in glioma development and progression. Several studies have correlated increased tumor vascularization with impaired patient survival [10, 11]. Although there are a variety of factors that promote angiogenesis, the main one is VEGF (vascular endothelial growth factor) a regulator of endothelial cell proliferation and capillary hyperpermeability involved in malignant glioma.

Preclinical reports strongly suggest that antiangiogenic therapy can have a clinical benefit due to a vascular density reduction of tumors leading to a decrease in tumor size [12, 13]. However, the current antiangiogenic therapies have shown only a moderate clinical efficacy, with poor results in advanced glioblastoma.

Regarding the other current research focus in this field, one possible agent for selectively triggering GBM tumor cell death is Mifepristone, which is a compound with activity as a progesterone and glucocorticoid receptor antagonist. Consequently, the use of Mifepristone for the inhibition of tumor cell growth has shown positive results principally in hormone-dependent (e.g., breast, prostate and ovarian) cancer.

Recently it was reported that vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), cyclin D1 and progesterone receptor (PR) expression levels are elevated in patients with high–grade astrocytomas and progesterone regulates astrocytomas growth through its interaction with progesterone receptor [14]. The same authors also demonstrated that progesterone increased VEGF and EGFR expression, and cell proliferation in two human astrocytoma cell lines derived from tumors of different evolution grades (U373 grade III and D54 grade IV) and these effects were inhibited by Mifepristone [14, 15]. Other authors have also showed that the expression of VEGF in prostate cancer cells DU 145 and PC3 treated with 10 micromol/L of Mifepristone was significantly decreased [16].

As a chemosensitizing agent, Mifepristone has been used to modulate the cytotoxic activity of doxorubicin [17], paclitaxel [18] and cisplatin [19].

We previously reported that Mifepristone was able to enhance the citotoxicity of cisplatin in cervical cancer cells in vitro and in vivo by increasing the intracellular as intratumoral concentration of cisplatin [20]. In another study using cervical cancer cells [21], we showed that the combination of cisplatin with a pure antiestrogen, ICI 182,780, induced the arrest of the cell cycle at the G2/M phase. The failure of this control checkpoint is believed to lead to genomic instability, resulting in hypersensitivity to radiation. However, the role of Mifepristone, alone or combined with other drugs, has been poorly studied as a chemo-radio-sensitizer.

The aim of the present study was to investigate whether Mifepristone can modulate the growth of glioma xenotrasplants treated with temozolamide and radiation, and/or decrease of VEGF expression.

After treatment of glioma xenografts with chemo-radiotherapy, tumor growth was evaluated (Figure 1). The tumor volume from control animals is showed up to day 17. Due to the large size of tumors, animals were sacrificed on that day. The statistical analysis shows no differences in tumor growth during the first 10 days, but starting from day 15 there was indeed a significant decrease in this value (p < 0.05) between the control and all experimental groups. At day 17 the reduction in tumor volume fluctuated between 2.5-fold, (Control vs. Irradiation alone: Rad) and 7-fold (Control vs. the combination of both chemotherapeutic agents and irradiation: Rad + Tmz + Mife). At day 25, the statistical analysis indicates that tumor volume of Rad + Tmz + Mife group was significantly less from that of the Rad group (Power = 0.84) and Temozolamide group (Rad + Tmz) (Power = 0.84), but not from than of the Mifepristone group (Rad + Mife). Statistical analysis also indicates a significantly lesser tumor growth in the Rad + Mife group than the Rad group (Power = 0.95). However, no difference was found in tumor growth between Rad vs. Rad + Tmz, or between Rad + Tmz vs. Rad + Mife.

Figure 2 shows tumor PET/CT image of a representative animal from each of the Rad, Rad + Tmz, Rad + Mife and Rad + Tmz + Mife groups. Left images are baseline at the beginning of treatments, and right images are at day 25. Blue indicates ¹⁸F-FDG uptake, red arrows indicate tumor location at baseline and day 25; green arrows show sites of typical ¹⁸F-FDG uptake in brown adipose tissue (BAT) in the neck of the mice. The physiological ¹⁸F-FDG uptake in areas of supraclavicular fat has recently been recognized as ¹⁸F-FDG uptake in brown adipose tissue (BAT) using PET/CT technology. BAT functions as a thermogenic organ by producing heat to maintain body temperature in mammals, and it is generally in deep cervical regions including the supraclavicular areas. BAT is known to exhibit increases glucose uptake when the sympathetic nervous system is activated by cold stimulation [22]. Accordingly, the anesthesia used in the animals subjected to PET/CT assays can decrease the temperature slightly at the moment of imaging. In our study, due to fact that the anesthesia used appeared to decrease the temperature at the moment of imaging, we observed a typical ¹⁸F-FDG uptake in BAT in the neck of animals.

Images show the changes in metabolic activity after treatments. Metabolic tumor volume, represented in terms of the VOI [23], is depicted in Figure 3. There was a significant difference between the VOI from the Rad group and that of the groups with combined chemo-radiotherapy treatments.

The toxicity of treatments is shown in the Figure 4. No change in weight was observed, indicating no systemic toxicity with any of the treatments (radiotherapy alone or combined with chemotherapy). Although there was light weight loss at the beginning of the combination treatment with irradiation plus Mifepristone and Temozolamide, the weight of the animals in this group returned to the pretreatment values by the end of the study.

Figure 5 shows the analysis of VEGF expression levels in tumors at the end of the study. The results indicate that compared to the control tumors, there was a significant decrease in this value after all treatments (radiotherapy alone or radiotherapy combined with Mifepristone and/or Temozolamide). No difference was observed between any of the groups with treatments, demonstrating that ionizing radiation alone or its combination with any of the tested chemotherapy agents caused a marked reduction in VEGF expression.

To date, there is no effective therapy for GBM, a highly aggressive tumor whose treatment has been a permanent challenge. The current treatments of high-grade glioma patients with the combination of radiation and chemotherapy can result in substantial lymphopenia, immunosuppression and opportunistic infections compromising patient survival [24]. Therefore, new alternatives are needed in order to increase patient survival and avoid recurrence.

We observed significant differences in tumor volume growth between the control and treated groups (Figure 1). Moreover, when Mifepristone was administered concomitant with radiation, tumor growth rate was slower than with the radiation alone. Furthermore, the reduction of tumor growth-rate was even more evident when we added Mifepristone to the Temozolamide-Radiation scheme. This suggests that Mifepristone can play an important role in the chemo-radio-sensitization of GBM xenografts.

PET/CT images (Figure 2) illustrate the effect of the different treatments, showing not only differences in tumor size at day 25 but also variations in ¹⁸F-FDG uptake in tumors, denoted by the blue-green color in the tumor mass. The later is better visualized in terms of the VOI values measured in the metabolic images from PET (Figure 3); as mentioned before, the VOI measures the volume concentration of ¹⁸F-FDG in the tumor and represents its metabolic activity. In this experiment we observed that the size of the VOI (in cm³ or mm³) is not always equal to the anatomical volume of the tumor (compare ordinate-scale in Figures 2 and 3), as result of the existent necrosis in some tumors (gray regions in tumors from Rad and Rad-Tmz groups at day 25 in Figure 2) where no uptake is observed. For this reasons the VOI should be used as a more representative way to evaluate the therapeutic response in tumors under treatment. Our group is working in this topic by performing some comparative studies between the simple measurement of the dimension of the tumor and the VOI.

The Mifepristone dose used in our study was chosen in accordance with previous reports in which a tumor growth rate inhibition in ovarian and prostate xenografts was observed [25, 26]. However, the total accumulated dose used in the present study was lower compared to that used in any of the reports in the literature. Interestingly, when patients with non-resectable meningioma were chronically exposed to Mifepristone, no severe side effects were observed [27]. Thus Mifepristone can be administered for prolonged periods. In addition our results demonstrated no significant change in the body weight of animals, suggesting that Mifepristone could be administered at higher doses.

Previous studies have shown that Mifepristone induces G1-S blockage of the cell cycle through inhibition of cdk2 activity in human ovarian cancer cells [25]. A reduction in cdK2 activity has been associated with an inhibition of the transcription factor E2F1, which modulates S-phase progression [28, 29]. Accordingly, the induced chemo-radiation-damage found in the present study might owe itself to the capacity of Mifepristone to arrest the G1-S Phase of the cell cycle.

Another possible mechanism involved in the sensitizer effect of this antihormonal agent is its antagonist action on progesterone and glucocorticoid receptors. It is widely accepted that progesterone participates in the development of different types of cancer as a transcription factor [14, 30, 31]. It was reported [14, 15] that progesterone-receptor isoforms are expressed in two cell lines (U373 and D54) derived from high-grade human astrocytoma. The same authors also demonstrated that progesterone increased VEGF and EGFR expression and cell proliferation, Mifepristone was able to inhibit not only the progesterone effects but also when it was administered alone significantly reduce astrocytoma cell growth in vitro.

It was reported that Mifepristone binds strongly to glucocorticoid receptors, being its binding affinity for these receptors approximately five and three times greater than progesterone and dexamethasone, respectively. Since the glioma C6 cells used in our study have an elevated expression of glucocorticoid receptors, the blockage of these receptors by Mifepristone probably led to the receptor transactivation inhibition and therefore the inhibition of cell proliferation.

Considering the extensive evidence that glioma cells produce high levels of VEGF, and Mifepristone decreases the expression of VEGF in prostate cancer cells [16], breast cancer [32] and gastric cancer cells [33]; we also investigated the possible participation of Mifepristone in the inhibition of VEGF expression in glioma xenografts.

It was reported that the use of antiangiogenic therapies in high-grade gliomas results in VEGF inhibition, improving the vascular function and tumor oxygenation that can increase the response to radiation [34].

To elucidate whether the treatment with radiation-Mifepristone-Temozolamide acted on angiogenesis, we evaluated the VEGF expression in GBM xenografts at end of the experiments. However, radiation alone was enough to drastically decrease VEGF production, so in our experimental conditions was not possible to prove the direct participation of Mifepristone on VEGF down-regulation (Figure 5). Therefore, in future studies will be necessary to performed different schemas of the treatments that demonstrate the influence of Mifepristone on VEGF expression.

More studies should be performed to understand the mechanism by which Mifepristone acts, either by itself or in combination with radiation and other drugs, to inhibit tumor growth. Tieszen CR et al. [35] reported that growth inhibition of cancer cells by antiprogestin Mifepristone is not dependent upon expression of nuclear progesterone receptors. They showed that Mifepristone is able to inhibit the growth of in vitro cancer cells derived from the nervous system, breast, prostate, ovary, and bone, with an absence of expression of classic nuclear progesterone receptor in nearly all these cells.

Consequently, the potential action of Mifepristone in chemo-radiation treatments of different tumors may be mediated by other mechanisms, including its participation in apoptosis, cell cycle arrest, and expression of ATM or other radiosensitizer proteins, mechanisms that have been observed in other cell types and therefore may also be contributing to the reduction in size of glioblastoma xenografts found presently (Figure 1).

The present study suggests several possible mechanisms for the significant decrease in GBM tumor size found with the addition of Mifepristone to the treatment with radiation or radiation plus temozolamide. Whatever the possible mechanism, the current results strongly suggest the potential of Mifepristone as a chemo-radio-sensitizer for the standard treatments of GBM tumors, for which currently available treatments have shown limited effects.

Future studies are necessary to explain the mechanisms related to the chemo-radio-sensitizing effect of Mifepristone in GBM, not only on tumor xenografts but also in ortotopic models of glioma.