Karenitecin ( bnp 1350 ) and flavopridol as radiosensitizers in malignant glioma

The poor prognosis of malignant glioma patients highlights the need to develop low toxicity, tumor specific agents with the ability to synergize with proven efficacious treatment modalities, e.g., ionizing irradiation. This paper investigates the potential of BNP1350 (karenitecin), a topoisomerase I-targeting anticancer agent, and flavopridol a cyclin-dependent kinase inhibitor as radiosensitizers at clinically relevant doses in glioblastoma cell lines. A clonogenic survival and apoptosis assays were performed to test the effect of karenitecin (0.1 nM to 10 nM), flavopridol, (50 nM to 500 nM), radiation (1 Gy to 5.5 Gy) and a combination of radiation and karenitecin or radiation and flavopridol on the glioma cell lines T986 and M059K. Cells were stained for cyclins B and D using antibodies followed by flow cytometry. Propidium Iodide staining was used to reveal the various phases of the cell cycle; cyclin staining in the G0/G1 and G2/M phase of the cell cycle was estimated as the Mean Fluorescence Intensity (MFI) after subtracting the MFI recorded by the isotype controls. Results demonstrated that in irradiated cells, pretreatment with karenitecin induced apoptosis, a transient arrest in the G2/M phase of the cell cycle and increased the expression of cyclin B1. Flavopridol treatment also induced apoptosis and a transient block in the G2/M phase of the cell cycle. The combined effects of karenitecin and flavopridol displayed synergistic effects. The unique radiosensitizing activity of orally administrable karenitecin and flavopridol is consistent with continued investigation of these compounds preclinically, as well as in the clinical setting. Karenitecin (bnp1350) and flavopridol as radiosensitizers in malignant glioma Deepika Rajesh, H. Ian Robins, Steven P. Howard K4 CSC, 600 Highland Avenue, University of Wisconsin Paul P Carbone Comprehensive Cancer Center, Madison, WI 53792, USA


Introduction
Surgery and radiotherapy currently remains the mainstay of treatment for high-grade gliomas. Advances in the last decade have included technological advances in both of these modalities, as well as the introduction of chemotherapy, e.g., temozolomide. Interestingly, the vast majority of recurrences (in radiation treated patients) will ultimately develop immediately within or in proximity to initial treatment fields. Thus, the development of an effective radiosensitizer is particularly relevant in the management of this poor prognosis patient group. As described below we elected to investigate two potential targets for inducing radiosensitization, i.e., topoisomerase1 and CDK. In selecting suitable inhibitors, parameters included potential clinical tolerability, and ability to cross the blood brain barrier (BBB).
Relative to this, cell cycle progression is controlled by the activity of CDKs, which in turn are regulated by cyclin accumulation, by positive and negative phosphorylation events, and by association with inhibitory proteins 11,12 . Two major classes of CDK inhibitors have been identified in mammalian cells and are referred to as the INK and KIP families 13 . The INK family member p16 INK-4a blocks progression through G 1 by inhibiting cyclin D kinases and plays a crucial role in cellular senescence [13][14][15] . The KIP-type inhibitors p21 WAF-1 and p27 Kip-1 regulate the progression from G 1 into the S phase by inhibiting cyclin E kinase activity. p21 WAF-1 is instrumental in invoking a G 1 arrest following DNA damage or perturbation of nucleotide metabolism 13 . Regulation of CDKs is altered in a number of tumor types, and therefore competitive CDK inhibitors targeting specifically to the ATP binding cleft are a particularly attractive target group of drugs that could be used in cancer treatment.
In summary, flavopiridol is a synthetic flavone, which has previously been evaluated as an antineoplastic agent [6][7][8][16][17][18][19][20][21] . It is a well-tolerated drug; the dose used in clinical trials ranges between 0.1-0.2 mg/ml. It induces growth arrest at either the G 1 and/or G 2 phases of the cell cycle in numerous cell lines in vitro by acting as a competitive binding agent for the ATP-binding pocket of CDK 22,23 . Flavopridol has been reported to bind to duplex DNA 1,24 . Flavopridol also inhibits receptor tyrosine kinases (EGFR), tyrosine kinases (pp60 Src) and signal transducing kinases (PKC and Erk-1) 23,25 . Although the inhibiting activity of flavopridol is strongest for CDK, the cytotoxic activity of flavopiridol is not limited to cycling cells as resting cells are also killed.

Karenitecin
Mammalian DNA topoisomerase I is the target of a number of active anticancer drugs known as camptothecins (e.g., topotecan and irinotecan). These topoisomerase inhibitors exert their cytotoxic effect by producing enzymemediated DNA damage, rather than by directly inhibiting enzyme catalytic activity. Recently, a series of novel camptothecin analogues, 7-silylcamptothecins ("silatecans"), have shown promising regression of U87 glioma cells in a nude mouse model and displayed lipophilicity to favor (BBB) transit 26 . Karenitecin a drug in this class (which has entered clinical trials) is a highly lipophilic, poorly watersoluble semisynthetic derivative of camptothecin, which can be administered orally. It displays increased stability at physiologic pH and has demonstrated cytotoxicity against human head and neck carcinoma and colon cancer cell lines 27,28 . The anti-tumor activity of karenitecin has been comparable of that of Topotecan in a xenograft model 29,30 . Grossman et al (2008) 31 have concluded a phase 1 study in recurrent glioma patients with a maximum tolerated dose of 1.5 mg/m 2 (and 2.0 mg/m 2 in patients receiving enzymeinducing anti-seizure drugs). The drug was well tolerated on a schedule of intravenous administration over 60 minutes daily for 5 days every 3 weeks. Median survival time after entering the study was 6.0 (95% CI 3.9 -9.7) months for 30 evaluable patients (23 glioblastoma; 7 anaplastic glioma).
The text that follows summarizes a preclinical investigation regarding the potential application of karenitecin and/or flavopridol as an adjunct to radiation treatment in malignant glioma cell lines.

Cell lines
The T98G 32,33 and MO59K 34 were obtained from ATCC and maintained in a humidified incubator with 5% CO 2 at 37C. T98G and MO59K cells were grown in DMEM F12 medium containing 10% fetal bovine serum, 1% penicillin, streptomycin and 1mM non-essential amino acids.

Cell treatment and clonogenic survival assays
The clonogenic survival assay was performed to test the effect of different doses of karenitecin (0.1nM to 10nM), flavopridol, (50nM to 500nM), radiation (1 Gy up to 8.5 Gy) and a combination of radiation and karenitecin or radiation and flavopridol on glioma cell lines. Stock solution of karenitecin was made in DMSO, and stock solution of flavopridol was made in PBS for all the experiments. Sub-confluent plates of glioma cell lines were treated with varying doses of karenitecin/flavopridol for a period of 72 hours. All cells were irradiated at a dose rate of approximately 7.5 Gy/min using a 137 Cs irradiator. The dose of irradiation varied from 1 Gy up to 8.5 Gy. Cells treated with different doses of radiation or DMSO [<0.1%] served as control for karenitecin treated cells, while cells treated with different doses of radiation or medium alone served as control for karenitecin treated cells. Following irradiation, both drug treated and untreated cells were harvested; washed with PBS; plated at the desired cell number and incubated for two weeks. The colonies were stained with crystal violet and percentage survival was quantified.
Survival was determined as the ratio of plating efficiencies for each irradiated group to that of the unirradiated control. For the flavopridol karenitecin combination experiments, the cells were pretreated with the indicated doses of either flavopridol or karenitecin; or a combination of both the drugs together for 72 hours followed by irradiation at 5.5 Gy. The cells were harvested, plated and the percentage survival was determined as described before.

Apoptosis assays
The percent of cells undergoing apoptosis and the different phases of the cell cycle were quantified by the method described by Darzynkiewicz et al 35,36  At appropriate times after treatment, the cells were harvested by trypsin-EDTA treatment and washed twice with ice-cold PBS. The cell pellet was resuspended in PBS and fixed in ice-cold 95% ethanol. The fixed cells were stained using a PI staining solution containing 20 ug/ml PI, 200 ug/ml RNase H and 0.1% NP40 solution. The cells were filtered through a 40µM pore nylon mesh (Tetkop, Inc.) and analyzed using a Beckton-Dickinson FACStar plus flow cytometry with excitation 488nM. The percentage of cells in sub-G 0 /G 1 peak and the distribution of cells in G 0 / G 1 , S and G 2 /M phases were determined using the Modfit LT version 2.0 (Verity Software, Topham, Maine) and Cell Quest software (Becton Dickinson, San Jose, CA).
A clonogenic survival assay was performed to test the effect of different doses of karenitecin (0.1 nM to 10 nM), flavopridol, (50 nM to 500 nM), radiation (1 Gy up to 8.5 Gy) and a combination of radiation and karenitecin or radiation and flavopridol on glioma cell lines 37 . Cell lines were treated with varying doses of karenitecin, or flavopridol for the indicated times, harvested by using cell dissociation solution, washed and subsequently stained with antibody to annexin V conjugated to FITC and PI [10 µg/ml] using the Apoalert Kit (Clonetech, Palo Alto, CA) according to the manufacturer's instructions. Viable [annexin V¯/PI¯] preapoptotic [annexin V + /PI¯], apoptotic [annexin V + /PI + ], and the residual damaged [annexin V¯/PI + ] cells are quantified using this protocol.

Staining for Cyclins
Staining for cyclins was performed according to the method described by Darzynkiewicz et al 38 . Briefly, subconfluent cultures of T98G and MO59K cells were treated with the indicated concentrations karenitecin in the presence and absence of radiation for 72 hours. The cells were harvested and fixed in ice-cold ethanol overnight at 4° C. The cells were washed, centrifuged, and suspended in permeabilization solution (0.25%v/v Triton X-100 in PBS pH 7.4) and kept on ice for 5 minutes. The cells were washed again, resuspended in 100μl wash buffer (1% BSA in PBS); and incubated with antibodies to cyclins B, D or isotype control (Santa Cruz biotechnology, CA) for 60 minutes. The cells were washed, and stained with goat anti-mouse FITC for 30 minutes at RT. The stained cell preparation was stained with 5 µg/ml PI and 200 µg/ml DNase-free RNase A. The cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and the results were acquired using CELL quest software and the Modfit LT version 2.0 (Verity Software, Topham, Maine). PI staining was used to reveal the various phases of the cell cycle and the cyclin staining in the G 0 /G 1 and G 2 /M phase of the cell cycle was estimated as the MFI after subtracting the Mean Fluorescence Intensity recorded by the isotype control for each sample.

Karenitecin induced radiosensitization in glioblastoma cell lines
The clonogenic survival assay was performed to determine the effect of different doses of karenitecin (0.   Treatment of T98G and MO59K cells with varying concentrations of karenitecin in the presence and absence of radiation for 72 hours revealed an increase in the G 2 /M phase of the cell cycle, and a decrease in the G 0 /G 1 phase of the cell cycle ( Figures 5 and 6). The increase in the G 2 /M phase of the cell cycle was dose dependent and also seen in the presence of radiation.

Karenitecin-treatment increased the levels of cyclins B and D
Distinct cyclins perform different tasks in specific phases of the cell cycle by binding to, and activating different cyclin dependent kinases. Cyclin B promotes G 2 /M transition, while the D-type cyclins (cyclin D1, D2, and D3) promote cell cycle progression from G 1 to S phase 39 . Since karenitecin induced a block in the G 2 /M phase of the cell cycle and a decrease in the G 0 /G 1 phase of the cell cycle, we evaluated karenitecin treated glioma cells for the presence of cyclin D1 and B1 in the G 0 /G 1 and G 2 /M phases of the cell cycle by flow cytometry. The results in Figure 7 revealed an increase in the levels of cyclin B1 predominantly in the G 2 /M phase of the cell cycle. This result correlated with the increase in the percentage of cells in the G 2 /M phase of the cell cycle following karenitecin treatment in both the cell lines. There was either no significant change or a slight decrease in the levels of cylcin D in cells present in the G 0 /G 1 phase of the cell cycle ( Figure 8). This finding correlated with the decrease in the percentage of cells in the G 0 /G 1 phase of the cell cycle. A slight increase in the expression of cyclin B1 was seen in the G 0 /G 1 phase of the cell cycle and an increase in the expression of cyclin D was also observed in the G 2 /M phase of the cell cycle, which could be attributed to the unscheduled cyclin production in glioma cells (Figure 8).

Flavopirdol induced radiosensitization in T98G cells
Since the onset of apoptosis by karenitecin was accompanied by an arrest in the G 2 /M phase of the cell   In order to determine the time of pretreatment needed for the radiosensitization to occur, T98G cells were treated with 200 nM of flavopridol between 2-72 hours. At the end of each pretreatment the cells were harvested and the surviving fraction was determined. The results showed that exposure to flavopridol for 8 hours was sufficient to cause radiosensitization ( Figure 10).

Flavopridol induced apoptosis accompanied changes in cell cycle progression
The next set of experiments was designed to determine the mode of death induced by flavopridol. Sub-confluent cultures of T98G cells were treated with flavopridol (50    karenitecin and radiation were clinically relevant. 72 hours was chosen as it caused the accumulation of cells in the G 2 /M phase of the cell cycle. A combination of karenitecin and flavopridol increased cell death greater than the additive effects caused by each of the drugs alone. This effect was further enhanced in the presence of radiation ( Figure 12). Both flavopridol and karenitecin alone or in combination caused an increase in the G 2 /M phase of the cell cycle (Figure13). These results demonstrate the ability of karenitecin and flavopridol to induce radiosensitization in glioma cells and indicate the ability of karenitecin to synergize with CDK inhibitors.

Discussion
Knowledge concerning the regulatory molecules involved in the control of growth and malignant progression is an essential component to conceptualization of effective agents for treatment of malignant glioma. The efficacy of many antitumor agents is dependent on DNA replication and cell cycle progression. Our results also reveal an increase in the cyclin D levels in glioma cells treated with both karenitecin and radiation. Over expression of cyclin D1 has been reported to alter sensitivity toward ionizing radiation in breast cancer cells 40,41 ; this could explain the increased levels of cyclin D in cells treated with both karenitecin and radiation. Karenitecin has been shown to induce apoptosis in head and neck cancer cell line A253 and cause growth inhibition of xenografts derived from childhood and adult highgrade glioma, medulloblastomas, ependymomas, as well as sub-lines resistant to procarbazine and busulfan by karenitecin 28,29 . This is the first report of the karenitecin as a radiosensitizer in glioma cells at clinically relevant doses of karenitecin and radiation.
Exposure to DNA damaging agents leads to growth arrest in the G 1 and G 2 phases of the cell cycle thereby preventing the cells from replicating and separating the damaged DNA. The growth arrest is due to activation of different cell cycle checkpoints surveillance proteins, which are activated by DNA damage or aberrant DNA replication. It is generally believed that the cell cycle arrest provides the cells with additional time to repair the potentially lethal DNA lesions, thereby rendering them more resistant to the cytotoxic effects of DNA damaging agents. Abrogation of the G 2 checkpoint is accompanied by increased cytotoxicity of many anticancer agents both in vitro and in vivo. The cyclin dependent kinase 2, cyclin B1 complex plays a key role in regulating the G 2 /M phase of the cell cycle. Although activation of the cyclin-dependent kinase CDC2 mediates G 2 /M transition in all tumor cells studied to date, regulation of CDC2 varies between tumor types. Persistent hyper phosphorylation of kinase and reduced cyclin expression have been implicated as mediators of treatment-induced G 2 delay in different tumor models.
Flavopridol is a potent inhibitor of most CDKs, including CDK1, CDK2, CDK4, and CDK7 9,23,42,43 . Multiple laboratories have shown flavopridol to induce apoptosis independent of the p53 and Rb status of the cell, promote differentiation, inhibit angiogenesis, modulate transcriptional events and block cell cycle progression via a transient G 2 /M block in the cell cycle. There have been reports on the synergistic effects of flavopridol in combination with other therapeutic drugs such as mitomycin, cytarabine, paclitaxel, and cisplatin [44][45][46] .
We have demonstrated that flavopridol could be a radiosensitizer at clinically relevant doses. Flavopridol induced apoptosis and a G 2 /M block in T98G cells. Flavopridol induced cell death was confirmed by dual colored (PI and Annexin V) flow cytometry (data not shown). Alonso et al 47 reported the ability of flavopridol to induce apoptosis in a panel of glioma cell lines via a caspase-independent mechanism. They also reported the downregualtion of MDM2 mRNA and protein levels and cyclin D 1 levels independent of Rb or p53 tumor suppressor pathway alterations. MDM2 protein regulates Rb and p53 function and under certain circumstances confers oncogenic properties 48 . Loss of MDM2 expression in tumor cells by flavopridol treatment could promote increased radiosensitivity.
Both kareniticin and flavopiridiol induce an arrest in the G 2 /M phase of the cell cycle. Their combined action affects cell replication and cell cycle regulation. Cells harboring mutated p53 (T98G) display a block in the G 2 /M phase of the cell cycle in response to Ionizing radiation indicating that the G 2 -phase block is a crucial event in the damage response of both these drugs and radiation. This G 2 -phase block is a crucial event in the damage response that can be manipulated to achieve a significant enhancement of drug toxicity potentially.
DNA might be a second target of flavopridol binding, as well as for karenitecin, and their combined effects could be also cytotoxic to non-cycling cancer cells.
In summary, we have shown, that orally administrable topoisomerase inhibitor karenitecin (BNP1350) and the cyclin dependent kinase inhibitor flavopridol both induce apoptosis, and radiosensitization of glioma cell lines. The radiosensitization appears to be independent of the p53 status since T98G cells have been demonstrated to harbor mutated p53. The combined effects of karenitecin and radiation or flavopridol with radiation or a combination of both the drugs with ionizing radiation could cause the shortening of the G 2 /M delay and cause sensitization of glioma cells to cytotoxic treatment.
Future studies aimed at understanding the underlying mechanisms of molecular radiosensitization with karenitecin and or flavopridol could provide valuable information for its further preclinical or clinical evaluation either as single agent or in combination with other agents in the treatment of malignant glioma.