Nalini Patel

Enhanced radioresponse of paclitaxel-sensitive and -resistant tumours in vivo

Paclitaxel is a potent chemotherapeutic drug and also has the potential to act as a radioenhancing agent. The latter is based on its ability to arrest cells in the radiosensitive G2M phases of the cell cycle; the weight of supporting evidence is derived mainly from in vitro studies. Our previous in vivo experiments identified enhanced tumour radioresponse predominantly attributable to tumour reoxygenation occurring as a result of paclitaxel-induced apoptosis. The current study investigated whether paclitaxel enhanced the radioresponse of tumours which are insensitive to apoptosis induction, but exhibited mitotic arrest, and compared the degree and kinetics of the response to that in tumours which develop apoptosis. The mouse mammary carcinoma MCa-29 (apoptosis sensitive) and the squamous cell carcinoma SCC-VII (apoptosis resistant) were used. In addition, the study investigated whether paclitaxel affected normal skin radioresponse to determine if a therapeutic gain could be achieved. Paclitaxel enhanced the radioresponse of both types of tumours. In the SCC-VII tumour, radiopotentiation occurred within 12 h of paclitaxel administration coincident with mitotic arrest, where enhancement factors (EFs) ranged from 1.15 to 1.37. In MCa-29 tumour, the effect was greater, EFs ranging from 1.59 to 1.91 and occurred between 24 and 72 h after paclitaxel when apoptosis was the predominant microscopic feature of treated tumours and when tumour oxygenation was found to be increased. The acute skin radioresponse and late leg contracture response were essentially unaffected by prior treatment with paclitaxel. Therefore, by two distinct mechanisms, paclitaxel was able to enhance the radioresponse of paclitaxel-sensitive and -resistant tumours, but not the normal tissue radioresponse, thus providing true therapeutic gain.

Relationship of Ki-67 labeling index to DNA-ploidy, S-phase fraction, and outcome in prostate cancer treated with radiotherapy

Our purpose was to evaluate the relationship of Ki‐67 labeling index (Ki67‐LI) to deoxyribonucleic acid (DNA) ploidy, S phase fraction (SPF), other clinical prognostic factors, and clinical outcome for patients with prostate cancer treated by external beam radiotherapy.

Combination of Anti-IGF-1R Antibody A12 and Ionizing Radiation in Upper Respiratory Tract Cancers

PURPOSE The IGF1/IGF-1R signaling pathway has emerged as a potential determinant of radiation resistance in human cancer cell lines. Therefore we investigated the potency of monoclonal anti-IGF-1R antibody, A12, to enhance radiation response in upper respiratory tract cancers. METHODS AND MATERIALS Cell lines were assessed for IGF-1R expression and IGF1-dependent response to A12 or radiation using viability and clonogenic cancer cell survival assays. In vivo response of tumor xenografts to 10 or 20 Gy and A12 (0.25-2 mg × 3) was assessed using growth delay assays. Combined treatment effects were also analyzed by immunohistochemical assays for tumor cell proliferation, apoptosis, necrosis, and vascular endothelial growth factor expression at Days 1 and 6 after start of treatment. RESULTS A12 enhanced the radiosensitivity of HN5 and FaDu head-and-neck carcinomas in vitro (p < 0.05) and amplified the radioresponse of FaDu xenografts in a dose-dependent manner, with enhancement factors ranging from 1.2 to 1.8 (p < 0.01). Immunohistochemical analysis of FaDu xenografts demonstrated that A12 inhibited tumor cell proliferation (p < 0.05) and vascular endothelial growth factor expression. When A12 was combined with radiation, this resulted in apoptosis induction that persisted until 6 days from the start of treatment and in increased necrosis at Day 1 (p < 0.01, respectively). Combined treatment with A12 and radiation resulted in additive or subadditive growth delay in H460 or A549 xenografts, respectively. CONCLUSIONS The results of this study strengthen the evidence for investigating how anti-IGF-1R strategies can be integrated into radiation and radiation-cetuximab regimen in the treatment of cancer of the upper aerodigestive tract cancers.

Zerumbone increases oxidative stress in a thiol-dependent ROS-independent manner to increase DNA damage and sensitize colorectal cancer cells to radiation

Locally advanced rectal cancers are treated with neoadjuvant chemoradiation therapy followed by surgery. In a minority (~20%) of patients, no tumor is present at the time of surgery; these patients with a complete pathologic response (pathCR) to neoadjuvant therapy have better treatment outcomes. Unfortunately, the inherent radioresistance of colorectal cancer (CRC) cells dictates that the majority of patients do not achieve a pathCR. Efforts to improve these odds have fueled the search for novel, relatively less‐toxic radiosensitizers with distinct molecular mechanism(s) and broad‐spectrum anticancer activities. Here, we use zerumbone, a sesquiterpene from the edible ginger (Zingiber zerumbet Smith), to enhance radiosensitivity of CRC cells. Short exposure to zerumbone (7 h) profoundly sensitized CRC cells, independent of their p53 or k‐RAS status. Zerumbone enhanced radiation‐induced cell cycle arrest (G2/M), increased radiation‐induced apoptosis, but induced little apoptosis by itself. Zerumbone significantly enhanced radiation‐induced DNA damage, as evident by delayed resolution of post‐irradiation nuclear γH2AX foci, whereas zerumbone treatment alone did not induce γH2AX foci formation. Zerumbone pretreatment inhibited radiation‐induced nuclear expression of DNA repair proteins ataxia‐telangiectasia mutated (ATM) and DNA‐PKcs. Interestingly, zerumbone‐mediated radiosensitization did not involve reactive oxygen species (ROS), but was mediated through depletion of cellular glutathione (GSH). Ability of only thiol‐based antioxidants to abrogate zerumbone‐mediated radiosensitization further corroborated this hypothesis. The α,β‐unsaturated carbonyl group in zerumbone was found to be essential for its bioactivity as zerumbone analog α‐Humulene that lacks this functional group, could neither radiosensitize CRC cells, nor deplete cellular GSH. Our studies elucidate novel mechanism(s) of zerumbones ability to enhance CRC radiosensitivity.

The effect of paclitaxel on the cell cycle kinetics of a murine mammary adenocarcinoma in vivo

Abstract: This article reports data that describe perturbations in cell‐cycle dynamic kinetic parameters following in vivo treatment of a syngeneic murine mammary tumor (MCa‐4) with paclitaxel. The kinetic parameters include the duration of the S (Ts) and G2M (TG2M) phases of the cell cycle, the tumor potential doubling time (Tpot), the bromodeoxyuridine (BrdUrd) labeling index (Ll), and the fraction of proliferating cells that successfully completed mitosis following paclitaxel treatment. When tumors transplanted into the right thigh of 4‐month‐old male C3Hf/kam mice grew to 8 mm mean diameter, the mice were injected intravenously with 40 mg/kg paclitaxel. At one of a range of times thereafter (1–72 hours) animals were pulse labeled with 60 mg/kg BrdUrd. Mice were sacrificed and tumors excised either immediately or at 3 or 6 hours after BrdUrd labeling. Single‐nuclei suspensions were prepared, and the kinetic parameters evaluated using bivariate DNA versus BrdUrd flow‐cytometric methods.

Abstract 3284: Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model

FLT3 internal tandem duplication (ITD) mutations are the most common alterations in acute myeloid leukemia (AML) and are associated with poor disease prognosis. Targeted therapy using FLT3-ITD inhibitors showed limited effect in reducing leukemia blasts in bone marrow (BM) than that in peripheral blood. The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents. Therefore, blocking CXCR4 and E-selectin concomitantly with FLT3 inhibition could theoretically eliminate the protection in FLT3-mutant AML patients. We recently reported that targeting CXCR4/E-selectin with the dual inhibitor GMI-1359 showed significant prolongation of survival of mice engrafted with FLT3-ITD mutant MV4-11 leukemia cells. In the present study, we further investigated anti-leukemia effects of dual CXCR4/E-selectin inhibition. Results indicate efficient mobilization of leukemia cells into the circulation by GMI-1359 in a MOLM14-engrafted murine model 2h after drug treatment, which was 3.3-fold (± 0.3) higher compared with the CXCR4 antagonist plerixafor, and 7.4-fold (± 2.7) higher compared with controls. In addition, GMI-1359 also mobilized normal murine leukocytes from the BM, suggesting that GMI-1359 may block interactions of leukemia cells with various BM components. Combination therapy of GMI-1359 and sorafenib significantly reduced leukemia burden (1.9e7 vs. 2.3e9, 1.0e9 and 8.5e7 photons/sec in the combination group versus control, GMI-1359 and sorafenib groups, respectively, at day 20 as determined by Xenogen IVIS bioluminescence Imaging; p Citation Format: Weiguo Zhang, Hong Mu, Qi Zhang, Nalini B. Patel, William E. Fogler, John L. Magnani, Michael Andreeff. Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3284.

Abstract 1161: Visualizing lung cancer heterogeneity by color-coding clonal cell subpopulations

Tumors are composed of heterogeneous cell populations exhibiting different biological features (e.g., growth rate, metastatic propensity, responsiveness to therapy) that differentially determine disease outcome. However, it has been difficult to map heterogeneity within tumors and correlate it to specific biological features. This study9s aim was to develop a model system where clonal tumor subpopulations are differentially color tagged and can be directly correlated with biological features. Two approaches were used to color-code tumor subpopulations: lung tumor H460 cells were stably transfected with either (a) fluorescence-tagged Histone H2b (GFP or RFP) plasmids or (b) Brainbow vectors encoding different fluorescent proteins with intervening loxP and loxP variant recombination sites. Cre induction generated stable, multicolor, clonal cell populations. When these cell populations were mixed and grown in vitro, each color-coded clone expanded and occupied a spatially distinct region discernable by color thresholding imaging techniques. When these same populations were injected into the leg of nude mice, they generated solid tumors exhibiting spatially distinct, color-coded neighborhoods of clonally derived cells. While the various populations exhibited similar cloning efficiencies in vitro, they exhibited different tumor growth capabilities and growth patterns (e.g., compact versus dispersed) in vivo. To further determine whether these clonal variants possess different biological behaviors, we tested their sensitivities to radiation using an in vitro clonogenic survival assay whereby mixed populations were irradiated with 2-6 Gy γ-rays and the identities of surviving clones determined by color thresholding. Color-coded clones significantly varied in their radiosensitivities (i.e., the radiation dose required to reduce survival to 0.1 ranged from 2.4- to 5-Gy, and the survival fraction at 2-Gy ranged from 0.15 to 0.91). While the most sensitive clone lacked a shoulder on the radiation dose-survival curve, the most resistant clone exhibited a broad shoulder indicative of high repair proficiency. The introduction of color did not affect radiation sensitivity. Established tumor xenografts of these mixed cell populations were exposed to 20-Gy radiation and then analyzed for preferential clonal regrowth over 12 days following radiation. Preliminary analyses revealed evidence of preferential clonal regrowth. These results suggest that this color-coding methodology offers a unique capability for visualizing clonal subpopulations within tumors and quantifying their impact on therapeutic outcome. Further molecular characterization (and genetic manipulation) of these subpopulations may facilitate delineation of critical factors determining behavior in heterogeneous tumors. (Supported in part by the Susan G. Komen BCRT0504146, NIH-NCI CA 06294, CA 103820, and DAMD W81XWH-04-1-01) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1161.