John M. Akudugu

Cytotoxicity of azadirachtin A in human glioblastoma cell lines.

The neem toxin azadirachtin A exhibits selective toxicity on insects. Despite its well-proven efficacy, the mode of action of this toxin remains obscure. The toxicity on vertebrate cells compared to insect cells is also not well characterized. We have cultivated six human glioblastoma cell lines G-28, G-112, G-60 (TP53 mutant) and G-44, G-62, G-120 (TP53 wild-type) in the presence of 28 microM of azadirachtin. This toxin concentration was chosen because it represents the 25 to 50% lethal dose in the glioma cells. Toxicity was measured in terms of cell proliferation (binucleation index), formation of micronuclei and cell survival. In the TP53 mutant cell lines, azadirachtin reduced the proportion of dividing cells and induced formation of micronuclei. Except for G-44 which showed a decrease in binucleation index, proliferation in the TP53 wild-type cell lines was unaffected by azadirachtin. In the TP53 wild-type cell lines, the decrease in micronuclei frequency is attributed to fewer cells entering mitosis to produce micronuclei. This is also apparent from the low surviving fractions. Cell survival was suppressed by 25-69% in all cell lines. The reduction of cell survival is a clear indication that azadirachtin affects reproductive integrity and cell division. The induction of micronuclei reflects DNA damage. Similar studies on damage induction in insect cell lines could elucidate the processes which precede the antifeedant and antimoulting effects of azadirachtin and other neem toxins in insects.

Frequency of Radiation-Induced Micronuclei in Neuronal Cells Does Not Correlate with Clonogenic Survival

Abstract Akudugu, J.M., Slabbert, J.P., Serafin, A. and Bohm, L. Frequency of Radiation-Induced Micronuclei in Neuronal Cells Does Not Correlate with Clonogenic Survival. It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2α, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2α, G28, G120 and G44; SF2 ≥ 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of 60Co γ radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2 ≤ 0.27) produced 0.08 ± 0.01 and 0.04 ± 0.01 MN per gray, respectively. OP-6, SK-N-BE(2c), G112, G62, G60 and KELLY cells constituted an intermediate group and displayed a micronucleus formation index between 0.10 and 0.24 MN per gray per binucleated cell. SHSY5Y cells showed no detectable formation of MN. In two groups [OP-6, SK-N-BE(2c), G112, G62, N2α and G28 or G120, G60, OP-27 and SK-N-SH], the more resistant cell lines produced more MN per unit dose. Another group [OP-6, SK-N-BE(2c), G112, G62, G44 and G120] showed no correlation between micronucleus formation and radiosensitivity. We conclude that the relationship between cell survival and micronucleus formation is not straightforward and that it would be simplistic to translate micronucleus frequency into radiosensitivity.

Changes in Lognormal Shape Parameter Guide Design of Patient-Specific Radiochemotherapy Cocktails

Uptake of radiopharmaceuticals and chemotherapeutic drugs is nonuniform at the microscopic level. Their distributions are typically lognormal, suggesting that failure in chemotherapy and targeted radionuclide therapy may be attributable, in part, to the characteristics of this biologically ubiquitous distribution. The lognormal problem can be overcome by using cocktails of 2 or more agents, tailored such that at least 1 agent is strongly incorporated by every cell in the target population. Therefore, critical assessment of the tissue uptake of each cocktail component is warranted. Methods: Cellular incorporation of the α-particle–emitting radiochemical (210Po-citrate) and 2 anticancer drugs (daunomycin and doxorubicin) was determined using flow cytometry. The role of their lognormal distribution in clonogenic cell survival was evaluated. Results: The shape parameter of the lognormal distribution was found to be correlated to both intracellular agent concentration and cell survival. Although no difference emerged between the shape parameters for citrate within the first 2 logs of cell kill, those for daunomycin and doxorubicin changed significantly. Conclusion: Changes in the value of the lognormal shape parameter and slope of the cellular drug uptake curves can be used to rapidly screen radiopharmaceuticals and other cytotoxic agents to formulate more effective cocktails for cancer therapy.

Influence of DNA double‐strand break rejoining on clonogenic survival and micronucleus yield in human cell lines

Purpose: To examine the role of DNA double‐strand break (DSB) rejoining in cell survival and micronucleus yield after 60Co γ‐irradiation. Materials and methods: Thirteen human cell lines (six glioblastoma, five prostate, one melanoma, one squamous cell carcinoma) were irradiated with 60Co γ‐rays to doses of 0–10 Gy for cell survival and micronucleus measurements and 0–100 Gy for DSB rejoining. Measurements were performed using standard clonogenic, micronucleus and constant‐field gel electrophoresis assays. Results: Radioresistance and micronucleus yield were positively correlated (r=0.74, p=0.004). A significant cell type‐dependent correlation was demonstrated between total (0–20 h) DSB rejoining and cell survival (r=0.86, p=0.03 for glioblastomas; r=0.79, p=0.04 for other cell lines), with more resistant cell lines showing higher levels of DSB rejoining. No relationship was apparent between fast (0–2 h) or slow (2–20 h) DSB rejoining and clonogenic survival. While there was no relationship between total or slow DSB rejoining and micronucleus yield, a significant and cell type‐specific correlation emerged between fast rejoining and micronucleus yield for the glioblastomas (r=0.89, p=0.04) and other cell lines (r=0.76, p=0.04). Cell lines with higher levels of DSB rejoining within 2 h of irradiation showed higher yields of micronuclei. Conclusion: Fast DSB rejoining, possibly through interaction with slow DSB rejoining, appears to play an important role in the formation of micronuclei. However, total DSB rejoining reflects intrinsic radiosensitivity. Consideration of differences in DSB rejoining kinetics might contribute to a better understanding of the significance of cell survival and micronucleus data in the clinical and radiation protection setting.

Flow cytometry-assisted Monte Carlo simulation predicts clonogenic survival of cell populations with lognormal distributions of radiopharmaceuticals and anticancer drugs.

Purpose: Although the distribution of therapeutic agents within cell populations may appear uniform at the macroscopic level, the distribution at the multicellular level is nonuniform. As such, the mean agent concentration in tissue may not be a suitable quantity for use in predicting biological effects. Failure in chemotherapy and targeted radionuclide therapy has been attributed, in part, to the ubiquity of lognormal distributions of therapeutic agents. To improve capacity to predict biological response, this work develops approaches that determine the fate of a cell population on a cell-by-cell basis. Methods: Incorporation of the α-particle emitting radiochemical (210Po-citrate) and two anticancer drugs (daunomycin and doxorubicin) by Chinese hamster V79 cells was determined using flow cytometry. Monte Carlo simulation was used to estimate cell survival on the bases of mean and individual cell incorporation of each cytotoxic agent. The interrelationships between the Monte Carlo simulated cell survival and clonogenic cell survival were evaluated. Results: Cell survival obtained by Monte Carlo simulation based on individual cell incorporation was in good agreement with clonogenic cell survival for all agents. However, the agreement was poor when the simulation was carried out using the mean cell incorporation of the agents. Conclusion: These data indicate that, with the aid of flow cytometry, Monte Carlo simulations can be used to predict the toxicity of therapeutic agents in a manner that takes into account the effects of lognormal and other nonuniform distributions of agents within cell populations.

A method to predict response of cell populations to cocktails of chemotherapeutics and radiopharmaceuticals: Validation with daunomycin, doxorubicin, and the alpha particle emitter 210Po

UNLABELLED There is considerable interest in the use of α-emitting radionuclides in radioimmunotherapy. However, the high toxicity of α-emitting radionuclides often does not permit administration of high activities for fear of normal tissue toxicity. Accordingly, targeting procedures need to be optimized for improved tumor control and minimized normal tissue toxicity. To guide design of effective cocktails of α-emitting radiopharmaceuticals and chemotherapy drugs, approaches that can predict biological response of a cell population on a cell-by-cell basis are needed. METHODS Cells were concomitantly treated with the α-particle emitting radiochemical (210)Po-citrate and daunomycin, or with (210)Po-citrate and doxorubicin. The responses of the treated cell populations were measured with a colony forming assay. The nonuniform cellular incorporation of the radiochemical and drugs was determined simultaneously on a cell-by-cell basis using flow cytometry. Monte Carlo methods were used to simulate cell survival on the basis of individual cell incorporation of each cytotoxic agent and validated by direct comparison with the experimental clonogenic cell survival. RESULTS Both daunomycin and doxorubicin enhanced the toxicity of the α-particles with a magnitude greater than expected based on single-agent toxicities. Cell survival obtained by Monte Carlo simulation was in good agreement with clonogenic cell survival for the combination treatments. CONCLUSION Flow cytometry assisted Monte Carlo simulations can be used to predict toxicity of cocktails of α-emitting radiopharmaceuticals and chemotherapy drugs in a manner that takes into account the effects of nonuniform distributions of agents within cell populations.

Micronucleus response of human glioblastoma and neuroblastoma cells toward low-LET photon and high-LET p(66)/Be neutron irradiation.

The identification of photon resistant tumors that are sensitive to neutrons is still an unresolved problem, and no radiobiological criteria have been developed that could help the selection of patients for neutron therapy. The micronucleus (MN) assay has been evaluated for this purpose in a panel of human glioblastoma and neuroblastoma cell lines spanning a wide range of photon sensitivities defined by mean inactivation doses (&OV0430;&ggr;) of 1.25–3.21 Gy. We show that the relative biologic effectiveness (RBE) of the p(66)/Be neutrons is significantly correlated with inherent photon sensitivity (r = 0.89, p < 0.01), indicating that the panel of cell lines used is suitable to study the differential biologic response to neutrons and photons. We find that p(66)/Be neutrons are 1.43 to 5.29 times more effective per unit dose in inducing micronuclei than 60Co &ggr;-rays. Surprisingly, cells that are inherently photon resistant tend to show a higher yield of micronuclei following exposure to either photons or neutrons, but no significant correlation could be demonstrated. However, RBE values based on micronucleus yield were found to strongly correlate with RBE values derived from cell survival data (r = 0.91, p < 0.01). It is concluded that although micronucleus yield does not reflect intrinsic sensitivity to either photons or neutrons, the strong correlation between RBE calculated from micronucleus formation and RBE derived from cell survival demonstrates that the micronucleus endpoint has a potential for detecting photon resistant cells that show increased sensitivity to neutrons.

The Advantage of Antibody Cocktails for Targeted Alpha Therapy Depends on Specific Activity

Nonuniform dose distributions among disseminated tumor cells can be a significant limiting factor in targeted α therapy. This study examines how cocktails of radiolabeled antibodies can be formulated to overcome this limitation. Methods: Cultured MDA-MB-231 human breast cancer cells were treated with different concentrations of a cocktail of 4 fluorochrome-conjugated monoclonal antibodies. The amount of each antibody bound to each cell was quantified using flow cytometry. A spreadsheet was developed to “arm” the antibodies with any desired radionuclide and specific activity, calculate the absorbed dose to each cell, and perform a Monte Carlo simulation of the surviving fraction of cells after exposure to cocktails of different antibody combinations. Simulations were performed for the α-particle emitters 211At, 213Bi, and 225Ac. Results: Activity delivered to the least labeled cell can be increased by 200%–400% with antibody cocktails, relative to the best-performing single antibody. Specific activity determined whether a cocktail or a single antibody achieved greater cell killing. With certain specific activities, cocktails outperformed single antibodies by a factor of up to 244. There was a profound difference (≤16 logs) in the surviving fraction when a uniform antibody distribution was assumed and compared with the experimentally observed nonuniform distribution. Conclusion: These findings suggest that targeted α therapy can be improved with customized radiolabeled antibody cocktails. Depending on the antibody combination and specific activity of the radiolabeled antibodies, cocktails can provide a substantial advantage in tumor cell killing. The methodology used in this analysis provides a foundation for pretreatment prediction of tumor cell survival in the context of personalized cancer therapy.

The potential of PAI-1 expression in needle biopsies as a predictive marker for prostate cancer

Abstract The relative abundance of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) in transurethral resections of the prostate (TURP) has been shown to correlate with disease state. The objective of this study was to assay for uPA and PAI-1 in prostate needle biopsies, and to test their potential as predictive markers for prostate cancer (PCa). uPA and PAI-1 levels were determined for 111 patients (55 PCa; 56 benign prostatic hyperplasia (BPH)), using the FEMTELLE enzyme-linked immunosorbent (ELISA) assay. The PAI-1 concentrations for PCa and BPH patients differed significantly (p = 0.0403) and a level of ≥ 4.5 ng/mg protein in men 60 years and older appears to be predictive of PCa, with a sensitivity of 63%. uPA plays a minor role as a potential marker in biopsy tissue, a feature noted in our recent TURP tissue studies, and elsewhere. The potential utility of the uPA/PAI-1 ratio as a predictor of prostate disease, as previously suggested for TURP tissue, is not apparent in needle biopsy tissue. PAI-1 concentration in prostate biopsies could be a candidate marker for distinguishing between PCa and BPH in older patients.

Strong synergism between small molecule inhibitors of HER2, PI3K, mTOR and Bcl-2 in human breast cancer cells

Targeting pro-survival cell signaling components has been promising in cancer therapy, but the benefit of targeting with single agents is limited. For malignancies such as triple-negative breast cancer, there is a paucity of targets that are amenable to existing interventions as they are devoid of the human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). Concurrent targeting of cell signaling entities other than HER2, PR and ER with multiple agents may be more effective. Evaluating modes of interaction between agents can inform efficient selection of agents when used in cocktails. Using clonogenic cell survival, interaction between inhibitors of HER2 (TAK-165), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) (NVP-BEZ235), and the pro-survival gene (Bcl-2) (ABT-263) in three human breast cell lines (MDA-MB-231, MCF-7 and MCF-12A) ranged from strong to very strong synergism. The strongest synergy was demonstrated in PR and ER negative cells. Inhibition of PI3K, mTOR and Bcl-2 could potentially be effective in the treatment of triple-negative cancers. The very strong synergy observed even at lowest concentrations of inhibitors indicates that these cocktails might be able to be used at a minimised risk of systemic toxicity. Concurrent use of multiple inhibitors can potentiate conventional interventions like radiotherapy and chemotherapy.