Telomerase is widely expressed in 80%–90% of human cancers, but it is almost undetectable in normal somatic cells.45
In human brain tissue, telomerase activity is observed in 89% of glioblastomas and 45% of anaplastic astrocytomas, but it is absent in normal brain tissue.46
In most cancers, the level of telomerase activity generally correlates with the proliferation state of the cells. The presence of the enzyme is required for unlimited proliferation (immortality), whereas its absence almost always dictates a finite lifespan (senescence).45
This suggests that telomerase activation is involved in the establishment of cellular immortality and may therefore be a critical step in carcinogenesis.47
Therefore, inhibiting telomerase activity may be a therapeutic strategy for selectively targeting malignant gliomas and sparing normal brain tissue.48
We examined the naturally occurring compound Bdph to investigate its possible antitumor activity in vitro and in vivo. We treated human glioblastoma cells with Bdph and observed a dose-dependent decrease in human telomerase reverse-transcriptase mRNA expression and a concomitant increase in p16 and p21 expression (data not shown). This was supported by data from a mouse xenograft model in which Bdph suppressed telomerase activity and inhibited tumor proliferation, which resulted in tumor senescence (data not shown).
Although telomerase is absent from most human somatic tissue, telomerase is expressed in adult germline tissues and in bone marrow hematopoietic cells. The presence of this telomerase activity may result in unwanted adverse effects if Bdph is systemically administered to treat malignant gliomas; this may pose a particular problem for long-term treatment regimens. In an attempt to solve this potential issue, we tested the local, controlled release of drugs bound to polymers. Overall, targeting the telomerase gene using a local delivery system to produce antitumor effects with drugs such as Bdph may result in a clinically applicable therapy in the future.
The carmustine implant, a currently used local delivery Gliadel wafer, is an alkylating agent that kills both tumor and normal cells. As a consequence, many adverse effects are observed even with local application. Adverse reactions are both local and mild (eg, nausea and vomiting) and systemic and aggravating (eg, infection, pulmonary embolus, and hemorrhaging). In contrast, systemic administration of Bdph results in only a mild hepatic impairment and slight hyperglycemia accompanying a 7.5 g/kg median lethal dose (LD50
). Their mild side effect is due to Bdph being a targeted drug. The effector of Bdph is the orphan receptor Nurr77, which translocates from the nucleus to the cytoplasm and leads to tumor apoptosis.23,24
In our previous study, we showed that Bdph increases Nurr77 transcription via an AP-1 motif.49,50
Nur77 has been implicated in growth inhibition and apoptosis,19,21,23,26–28,32
suggesting that Nur77 induction may be an early event during Bdph-induced apoptosis in GBM cells. To determine whether the same molecular mechanism is involved in the antitumor effect, we examined the effects of Bdph on tumor viability (Fig. ), signal transduction (Fig. ), Nurr77 protein translocation (Fig. ), and histochemical staining of in vivo brain tumor cell xenografts (Fig. ) when it is released from biodegradable wafers. Our results implicated the PKC pathway in signal transduction (Fig. A), showed Nur77 translocation from the nucleus to the cytoplasm (Fig. ), and demonstrated caspase expression leading to tumor cell apoptosis (Fig. C). These results are consistent with the effects of Bdph when it is not incorporated into polymer form. Our findings suggest that the Bdph-Wafer retains its natural biological antitumor function.
Sekiya et al. (2000) reported the biodistribution of Bdph. Less than 1 h after dermal application, labeled Bdph (unchanged [8-14
C]butylidenephthalide and/or its metabolite) was detected at the application site and in the liver, bile, and kidney. At 4 h, the labeled compound was still strongly detected in the intestines, although the total amount in the body decreased. The biodistribution data showed that only 0.029 μg/g of brain tissue was detected after 1 h.51
Thus, only 3% of the total amount can reach the brain. Although Bdph is a hydrophobic compound, the BBB most likely contributes to the low amount of Bdph found in the brain. Therefore, controlled local delivery of the drug to the target could circumvent this problem. We showed that approximately one-half of the available Bdph in the 10% Bdph-Wafer (~5 mg) was released from the degraded polymer at 6 days and that 99% was released by the 30th day. Thus, nearly 100% of the available Bdph reached the brain tumor using our biodegradable polymer.
Sekiya et al. (2000) also reported the pharmacokinetics of Bdph. After dermal application, the total radioactivity from labeled Bdph was decreased because of excretion into the urine. With intravenous administration, 80% of the administered Bdph was excreted into the urine within 24 h, whereas only 5% was excreted into the feces during this time.51
In contrast, detectable amounts of Bdph released from our local, controlled delivery system were maintained for up to 30 days (Fig. ). Our results demonstrate that the Bdph-Wafer not only led to an increase in the local compound concentration (ie, 50-fold) but also produced a concentration that remained stable for up to 30 days.
Finally, compared with the carmustine-Gliadel wafer, the Bdph-Wafer targets telomerase and therefore is expected to have no significant toxicity on surrounding normal brain cells. In addition, Bdph has a higher lethal dose than does carmustine (7500 mg/kg vs. 83 mg/kg), which encourages us to test higher concentrations and different formulations (such as glue or paste) as well as other biodegradable polymers (such as poly(lactic-co-glycolic acid), chitosan, and hydrogel) to increase the stability and local Bdph concentration.
Finally, to study the effect of the interstitial administration of Bdph against cranial brain tumors, FGF-SV40 transgenic mice were used. Our implantable biodegradable anhydride significantly reduced tumor size in a dose-dependent manner (Fig. ). There was no brain edema, no delay in wound healing, no CSF leakage, and no brain infection—all symptoms that have been observed for BCNU-Wafers.34,35
This may be due to the fact that Bdph is less toxic (LD50
, 7.5 g/kg) than BCNU (LD50
, 20 mg/kg). In addition, in the FGF-SV40 mouse model, spontaneous brain tumors are located in the frontal region of the cerebellum and posterior region of the fourth ventricle. Because our wafer was implanted at the back of the cerebellum, it would seem that the Bdph-Wafer can penetrate brain tissue to reach the tumor without injury to normal cells. As a result, we believe that the Bdph-Wafer would have an even greater effect on a tumor when it is in direct contact with the cavity wall after tumor removal. In addition, this treatment may be useful for GBMs that are present in the brainstem, a location that is difficult to reach surgically.
We have shown that the antitumor effect of Bdph is due to Nur77 translocation from the nucleus to the cytoplasm, leading to cytochrome c release and tumor apoptosis.19
According to a pharmacokinetic study, the half-life of Bdph is ~1 day, with excretion from urine.51
In this wafer-based system, Bdph is slowly released for up to 30 days (Fig. ). More importantly, at 30 days after implantation, Nurr77 translocation and tumor cell apoptosis were still observed in our nude mouse model (Fig. C). Thus, our wafer device maintains the stability and effectiveness of the Bdph compound for 30 days.
In summary, our study showed that polymers containing Bdph, a novel potential Nurr77-induced apoptosis drug, increase the local Bdph concentration and maintain its stability, leading to significant inhibition of tumor growth.