Purpose:Temozolomide, a DNA methylating agent, is currently undergoing clinical evaluation for cancer therapy. Because temozolomide has been shown to increase survival rates of patients with malignant gliomas when given combined with radiation, and there is conflicting preclinical data concerning the radiosensitizing effects of temozolomide, we further investigated the possible temozolomide-induced enhancement of radiosensitivity. Experimental Design: The effects of temozolomide on the in vitro radiosensitivity of U251 (a human glioma) and MDA-MB231BR (a brain-seeking variant of a human breast tumor) cell lines was evaluated using clonogenic assay. DNA damage and repair were evaluated using phosphorylated histone H2AX (gH2AX), and mitotic catastrophe was measured using nuclear fragmentation. Growth delay was used to evaluate the effects of temozolomide on in vivo (U251) tumor radiosensitivity. Results: Exposure of each cell line to temozolomide for 1 h before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1ranging from 1.30 to 1.32. Temozolomide had no effect on radiation-induced apoptosis or on the activation of the G 2 cell cycle checkpoint. As a measure of DNA double strand breaks, gH2AX foci were determined as a function of time after the temozolomide + irradiation combination. The number of gH2AX foci per cell was significantly greater at 24 h after the combined modality compared with the individual treatments. Mitotic catastrophe, measured at 72 h, was also significantly increased in cells receiving the temozolomide + irradiation combination compared with the single treatments. In vivo studies revealed that temozolomide administration to mice bearing U251 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay with a dose enhancement factor of 2.8. Conclusions: These results indicate that temozolomide can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair leading to an increase in mitotic catastrophe.Temozolomide has known anticancer effects against a broad range of tumor histologies including gliomas (1, 2). Temozolomide is a lipophilic molecule that can be given p.o. and crosses the blood-brain barrier. At physiologic pH, temozolomide is converted to the active metabolite methyltriazenoimidazole-carboxamide, which forms methyl adducts at O 6 -position of guanine in DNA. The formation of O 6 -methylguanine then results in mismatch pairing with thymine during subsequent cycles of DNA replication, followed by DNA strand-break formation and eventually cell death (2). Critical to its effectiveness as an antitumor agent is the cellular expression of O 6 -methlyguanine-DNA-methyltransferase (MGMT; refs. 3,4), which removes the O 6 -methyl adducts and, thus, acts to repair temozolomide-induced DNA damage (2, 3). In a recently published study by Stupp et al. (5), patients with primary glioblastoma multiforme (GBM) treated...

BackgroundGlioblastoma multiforme (GBM) is among the most lethal of all human tumors, with frequent local recurrences after radiation therapy (RT). The mechanism accounting for such a recurrence pattern is unclear. It has classically been attributed to local recurrence of treatment-resistant cells. However, accumulating evidence suggests that additional mechanisms exist that involve the migration of tumor or tumor stem cells from other brain regions to tumor bed. VEGFs are well-known mitogens and can be up-regulated after RT. Here, we examine the effect of irradiation-induced VEGF on glioma cell motility.Materials and methodsU251 and LN18 cell lines were used to generate irradiated-conditioned medium (IR-CM). At 72 h after irradiation, the supernatants were harvested. VEGF level in IR-CM was quantified by ELISA, and expression levels for VEGF mRNA were detected by RT-PCR. In vitro cancer cell motility was measured in chambers coated with/without Matrigel and IR-CM as a cell motility enhancer and a VEGF antibody as a neutralizer of VEGF bioactivity. Immunoblots were performed to evaluate the activity of cell motility-related kinases. Proliferation of GBM cells after treatment was measured by flow cytometry.ResultsIrradiation increased the level of VEGF mRNA that was mitigated by pre-RT exposure to Actinomycin D. U251 glioma cell motility (migration and invasion) was enhanced by adding IR-CM to un-irradiated cells (174.9 ± 11.4% and 334.2 ± 46% of control, respectively). When we added VEGF antibody to IR-CM, this enhanced cell motility was negated (110.3 ± 12.0% and 105.7 ± 14.0% of control, respectively). Immunoblot analysis revealed that IR-CM increased phosphorylation of VEGF receptor-2 (VEGFR2) secondary to an increase in VEGF, with a concomitant increase of phosphorylation of the downstream targets (Src and FAK). Increased phosphorylation was mitigated by adding VEGF antibody to IR-CM. There was no difference in the mitotic index of GBM cells treated with and without IR-CM and VEGF.ConclusionsThese results indicate that cell motility can be enhanced by conditioned medium from irradiated cells in vitro through stimulation of VEGFR2 signaling pathways and suggest that this effect involves the secretion of radiation-induced VEGF, leading to an increase in glioma cell motility.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in the USA with a median survival of approximately 14 months. Low survival rates are attributable to the aggressiveness of GBM and a lack of understanding of the molecular mechanisms underlying GBM. The disruption of signaling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. To identify protein kinases required for the survival of GBM we performed a siRNA-based RNAi screen focused on the human kinome in GBM. Inhibition of the polo-like kinase 1 (PLK1) induced a reduction in the viability in two different GBM cell lines. To assess the potential of inhibiting PLK1 as a treatment strategy for GBM we examined the effects of a small molecule inhibitor of PLK1, GSK461364A, on the growth of GBM cells. PLK1 inhibition arrested cells in the mitotic phase of the cell cycle and induced cell kill by mitotic catastrophe. GBM engrafts treated with GSK461364A showed statistically significant inhibition of tumor growth. Further, exposure of different GBM cells to RNAi or GSK461364A prior to radiation resulted in an increase in their radiosensitivity with dose enhancement factor ranging from 1.40 to 1.53 with no effect on normal cells. As a measure of DNA double strand breaks, γH2AX levels were significantly higher in the combined modality as compared to the individual treatments. This study suggests that PLK1 is an important therapeutic target for GBM and can enhance radiosensitivity in GBM.

NFκB is an inducible transcription factor that controls

The DNA double-strand break (DSB) is the primary lethal lesion after therapeutic radiation. Thus, the development of assays to detect and to quantitate these lesions could have broad preclinical and clinical impact. Phosphorylation of histone H2AX to form γ-H2AX is a known marker for irradiation-induced DNA DSBs. However, the first generation assay involves the use of immunofluorescent staining of γ-H2AX foci. This assay is time consuming, operator dependent and is not scalable for high throughput assay development. Thus, we sought to develop a new assay using a high throughput electrochemiluminescent platform from Mesoscale Discovery Systems to quantify γ-H2AX levels. The results show that our assay utilizes significantly less time and labor, has greater intra-assay reproducibility and has a greater dynamic range of γ-H2AX versus irradiation dose.

Objective: To report the incidence of chemotherapy-related amenorrhea (CRA) from chemotherapy with/ without adjuvant endocrine therapy in premenopausal women with breast cancer and to analyze the related factors.Design: From January 2000 to August 2006, 326 premenopausal women (e50 y old) who completed chemotherapy were available for analysis. The CRA definitional criterion in this study was no menstruation for 6 months in a woman who was premenopausal at diagnosis. As risk factors for CRA, woman's age, the type of chemotherapy regimen, adjuvant endocrine therapy use, and body mass index were evaluated.Results: The median age was 42 years (range, 22-50 y). The median follow-up period was 37 months (range, 12-80 mo). Women were divided into two groups by age at diagnosis: 128 women in group 1 (less than 40 years old) and 198 women in group 2 (age Q40 y). CRA occurred in a total of 223 (68%) women: 43% in group 1 and 85% in group 2 (P G 0.001). Despite CRA, 14% resumed menstruation: 24% in group 1 and 11% in group 2. Another 40 (12%) women had less than 6 months of menstruation interruption. Permanent CRA was related with age at diagnosis and use of adjuvant endocrine therapy (P G 0.05). In this study, there were four pregnancies, two of which resulted in therapeutic abortion due to ongoing chemotherapy.Conclusions: This study confirmed that the rate of CRA depends on age at diagnosis and the use of adjuvant endocrine therapy. It is essential to inform young women of reproductive age of the possibility of amenorrhea or resumption of menstruation and contraceptive options.

PurposeTo assess changes in oral cavity (OC) shapes and radiation doses to tongue with different tongue positions during intensity-modulated radiation therapy (IMRT) in patients with head and neck squamous cell carcinoma (HNSCC) but who refused or did not tolerate an intraoral device (IOD), such as bite block, tongue blade, or mouthpiece.ResultsTongue volume outside of OC was 7.1 ± 3.8 cm3 (5.4 ± 2.6% of entire OC and 7.8 ± 3.1% of oral tongue) in IMRT-S. Dmean of OC was 34.9 ± 8.0 Gy and 31.4 ± 8.7 Gy with IMRT-N and IMRT-S, respectively (p < 0.001). OC volume receiving ≥ 36 Gy (V36) was 40.6 ± 16.9% with IMRT-N and 33.0 ± 17.0% with IMRT-S (p < 0.001). Dmean of tongue was 38.1 ± 7.9 Gy and 32.8 ± 8.8 Gy in IMRT-N and IMRT-S, respectively (p < 0.001). V15, V30, and V45 of tongue were significantly lower in IMRT-S (85.3 ± 15.0%, 50.6 ± 16.2%, 24.3 ± 16.0%, respectively) than IMRT-N (94.4 ± 10.6%, 64.7 ± 16.2%, 34.0 ± 18.6%, respectively) (all p < 0.001). Positional offsets of tongue during the course of IMRT-S was –0.1 ± 0.2 cm, 0.01 ± 0.1 cm, and –0.1 ± 0.2 cm (vertical, longitudinal, and lateral, respectively).Materials and Methods13 patients with HNSCC underwent CT-simulations both with a neutral tongue position and a stick-out tongue for IMRT planning (IMRT-N and IMRT-S, respectively). Planning objectives were to deliver 70 Gy, 63 Gy, and 56 Gy in 35 fractions to 95% of PTVs. Radiation Therapy Oncology Group (RTOG) recommended dose constraints were applied. Data are presented as mean ± standard deviation and compared using the student t-test.ConclusionsIMRT-S for patients with HNSCC who refused or could not tolerate an IOD has significant decreased radiation dose to the tongue than IMRT-N, which may potentially reduce RT related toxicity in tongue in selected patients.

Although surgery is the mainstay of local treatment for skin cancer, definitive radiation therapy (RT) has been also applied for patients who are unable to tolerate surgery. Definitive RT regimens usually consist of daily treatment for 4–7 weeks. Such protracted daily RT regimens, however, would not be feasible for non-compliant patients or patients who are unable to make multiple daily trips for weeks. Without treatment, however, skin cancers can continuously progress and cause distressing symptoms. A cyclical hypofractionated RT (QUAD Shot: 14 Gy in 4 fractions, twice-daily treatments with 6 hours interval on 2 consecutive days) can be a practical RT regimen for those patients. In this report, we present the successful treatment course of repeated QUAD Shots in a 79-year-old patient with neglected skin cancer that was disfiguring his face yet declined definitive surgery and protracted RT. We also evaluated and compared biologically equivalent doses between QUAD Shots and conventionally fractionated protracted RT regimens.