Abstract and Introduction
Abstract
Object: The chemotherapeutic agent temozolomide has demonstrated antitumor activity in patients with recurrent malignant glioma. Because responses are not enduring and recurrence is nearly universal, further improvements are urgently needed.
Methods: In an effort to increase the clinical activity of temozolomide, the authors investigated whether its antitumor activity could be enhanced by adding tamoxifen or hypericin, two drugs that are known to inhibit the activity of protein kinase C. Human glioblastoma multiforme cell lines A172 and LA567 were treated with combinations of temozolomide and tamoxifen or hypericin in vitro, and cell survival was analyzed using various methods. Tamoxifen and hypericin were able to greatly increase the growth-inhibitory and apoptosis-stimulatory potency of temozolomide via the downregulation of critical cell cycleregulatory and prosurvival components. Furthermore, with the use of an in vivo xenograft mouse model, the authors demonstrated that hypericin was able to enhance the antiglioma effects of temozolomide in the in vivo setting as well.
Conclusions: Taken together, analysis of the results indicated that combination therapy involving temozolomide and tamoxifen or hypericin potently inhibited tumor growth by inducing apoptosis and provided an effective means of treating malignant glioma.
Introduction
Despite aggressive treatment, malignant GBM has remained difficult to treat, and its overall response to treatment has remained poor, as has outcome in patients harboring this lesion. Clearly, new treatment strategies are urgently needed. One of the more potent antiglioma chemotherapeutic agents with demonstrated efficacy is temozolomide (Temodar [in the US], Temodal [globally]; Schering Corporation, Kenilworth, NJ), an orally administered alkylating agent that is well absorbed and readily crosses the bloodbrain barrier. Meaningful anti-GBM activity of temozolomide has been demonstrated in clinical trials when used alone or in combination with other drugs such as adenovirally delivered tumor necrosis factorα, thalidomide, etoposide, irinotecan, or radiotherapy. However, although temozolomide is effective in delaying disease progression and maintaining health-related quality of life, durable responses have not yet been documented and the patients eventually die of this disease. To explore additional ways of increasing temozolomide-induced tumor-cell killing, we investigated whether its anti-GBM efficacy could be enhanced by two drugs that are known to inhibit the important cell survival enzyme, PKC.
It is known that highly malignant tumor cells can escape cell death by overexpressing various prosurvival factors, such as antiapoptotic proteins, repair enzymes, or various protein kinases that are involved in intracellular signal transductionsuch as PKC or mitogen-activated protein kinases. For example, increased activity of members of the PKC family has been implicated in cancer progression and is frequently found in malignant GBM cells compared with untransformed glial cells. High PKC activity correlates strongly with increased tumor cell proliferation and migration and provides a survival advantage because of PKC's ability to stimulate the synthesis of DNA repair enzymes and the antiapoptotic protein Bcl-2. In this regard, the authors of previous works have found that the growth rates of malignant GBM cells are exquisitely sensitive to modulation by PKC in vitro and that the use of PKC inhibitors to downregulate or directly inhibit PKC activity leads to reduced GBM cell proliferation and apoptosis. Additionally, PKC inhibitors have been found to enhance the cytotoxic effect of the topoisomerase inhibitor, Irinotecan, in GBM cells. Therefore, PKC inhibitors may act as chemosensitizers for GBM and potentiate the activity of other chemotherapeutic drugs.
In our work, we have used two different drugs, tamoxifen and hypericin, which are known to exert pleiotropic molecular effects, most notably the potent inhibition of PKC. Although tamoxifen is primarily used as an estrogen antagonist in the treatment of breast cancer, the authors of other studies have reported finding additional estrogen receptorindependent functions that involve the inhibition of various cellular components, including primarily PKC. When high-dose tamoxifen administration was investigated for use in recurrent high-grade gliomas, a 20 to 40% response rate was reported. In a small recently completed Phase II study of concurrent continuous tamoxifen and temozolomide for recurrent malignant astrocytic gliomas, no improvement was reported, although the authors noted that their study was perhaps too small to detect differences and that the selected doses and schedules were not optimized.
Hypericin is a naturally occurring photosensitizer found in Hypericum perforatum plants, commonly known as St. John's wort. The investigators of several studies have established its potent in vitro and in vivo antineoplastic activity, at least some of which apparently occurs in the absence of light activation. At the molecular level, it was found that hypericin-induced tumor cell death appears to involve several intracellular signaling pathways, including the inhibition of PKC and mitogen-activated protein kinases p42/p44. Several groups investigating GBM cell lines have demonstrated that this compound is able to exert potent antiglioma effects and to inhibit motility and invasion of GBM cells in vitro.
In consideration of the inhibitory effects of tamoxifen and hypericin on intracellular growth and survival pathways, we sought to determine whether these compounds might enhance the antitumor effects of temozolomide and could, thus, be considered when designing more effective regimens of anti-GBM therapy. In the present study we demonstrate that both of these drugs were able to greatly increase the antiproliferative and apoptosis-inducing efficacy of temozolomide on GBM cells in vitro. In addition, hypericin was able to significantly (p < 0.05) increase temozolomide's antitumor effect in a xenograft mouse model in vivo.