Brent W. Williams

Enhancement of tumor thermal therapy using gold nanoparticle–assisted tumor necrosis factor-α delivery

Tumor necrosis factor-α (TNF-α) is a potent cytokine with anticancer efficacy that can significantly enhance hyperthermic injury. However, TNF-α is systemically toxic, thereby creating a need for its selective tumor delivery. We used a newly developed nanoparticle delivery system consisting of 33-nm polyethylene glycol–coated colloidal gold nanoparticles (PT-cAu-TNF-α) with incorporated TNF-α payload (several hundred TNF-α molecules per nanoparticle) to maximize tumor damage and minimize systemic exposure to TNF-α. SCK mammary carcinomas grown in A/J mice were treated with 125 or 250 μg/kg PT-cAu-TNF-α alone or followed by local heating at 42.5°C using a water bath for 60 minutes, 4 hours after nanoparticle injection. Increases in tumor growth delay were observed for both PT-cAu-TNF-α alone and heat alone, although the most dramatic effect was found in the combination treatment. Tumor blood flow was significantly suppressed 4 hours after an i.v. injection of free TNF-α or PT-cAu-TNF-α. Tumor perfusion, imaged by contrast enhanced ultrasonography, on days 1 and 5 after treatment revealed perfusion defects after the injection of PT-cAu-TNF-α alone and, in many regions, complete flow inhibition in tumors treated with combination treatment. The combination treatment of SCK tumors in vivo reduced the in vivo/in vitro tumor cell survival to 0.05% immediately following heating and to 0.005% at 18 hours after heating, suggesting vascular damage–mediated tumor cell killing. Thermally induced tumor growth delay was enhanced by pretreatment with TNF-α-coated gold nanoparticles when given i.v. at the proper dosage and timing. [Mol Cancer Ther 2006;5(4):1014–20]

Metformin kills and radiosensitizes cancer cells and preferentially kills cancer stem cells

The anti-cancer effects of metformin, the most widely used drug for type 2 diabetes, alone or in combination with ionizing radiation were studied with MCF-7 human breast cancer cells and FSaII mouse fibrosarcoma cells. Clinically achievable concentrations of metformin caused significant clonogenic death in cancer cells. Importantly, metformin was preferentially cytotoxic to cancer stem cells relative to non-cancer stem cells. Metformin increased the radiosensitivity of cancer cells in vitro, and significantly enhanced the radiation-induced growth delay of FSaII tumors (s.c.) in the legs of C3H mice. Both metformin and ionizing radiation activated AMPK leading to inactivation of mTOR and suppression of its downstream effectors such as S6K1 and 4EBP1, a crucial signaling pathway for proliferation and survival of cancer cells, in vitro as well as in the in vivo tumors. Conclusion: Metformin kills and radiosensitizes cancer cells and eradicates radioresistant cancer stem cells by activating AMPK and suppressing mTOR.

Anginex synergizes with radiation therapy to inhibit tumor growth by radiosensitizing endothelial cells

We have demonstrated that the designed peptide anginex displays potent antiangiogenic activity. The aim of our study was to investigate the effect of anginex on established tumor vasculature as an adjuvant to radiation therapy of solid tumors. In the MA148 human ovarian carcinoma athymic mouse model, anginex (10 mg/kg) in combination with a suboptimal dose of radiation (5 Gy once weekly for 4 weeks) caused tumors to regress to an impalpable state. In the more aggressive SCK murine mammary carcinoma model, combination of anginex and a single radiation dose of 25 Gy synergistically increased the delay in tumor growth compared to the tumor growth delay caused by either treatment alone. Immunohistochemical analysis also demonstrated significantly enhanced effects of combined treatment on tumor microvessel density and tumor or endothelial cell proliferation and viability. In assessing physiologic effects of anginex, we observed a reduction in tumor perfusion and tumor oxygenation in SCK tumors after 5–7 daily treatments with anginex with no reduction in blood pressure. To test anginex as a radiosensitizer, additional studies using SCK tumors were performed. Three daily i.p. injections of anginex were able to enhance the effect of 2 radiation doses of 10 Gy, resulting in 50% complete responses, whereas the known antiangiogenic agent angiostatin did not enhance the radiation response of SCK tumors. Mechanistically, it appears that anginex functions as an endothelial cell‐specific radiosensitizer because anginex showed no effect on in vitro radiosensitivity of SCK or MA148 tumor cells, whereas anginex significantly enhanced the in vitro radiosensitivity of 2 endothelial cell types. This work supports the idea that the combination of the antiangiogenic agent anginex and radiation may lead to improved clinical outcome in treating cancer patients.

Nanotherapeutics for enhancing thermal therapy of cancer

Purpose: The current work describes the synergistic enhancement of hyperthermic cancer therapy by selective thermal sensitization and induction of vascular injury at the tumor site. The specificity of this response was mediated by CYT-6091: a pegylated colloidal gold-based nanotherapeutic designed to selectively deliver an inflammatory cytokine, tumor necrosis factor alpha (TNF), to solid tumors. Materials and methods: FSaII murine fibrosarcoma-bearing C3H mice received an intravenous injection of either soluble TNF or CYT-6091 (50–250 µg/kg TNF). Four hours later the tumors were exposed to localized heating (42.5 or 43.5°C, 60 min). Tumor responses were assessed by growth delay and/or perfusion. Results: Both soluble TNF and CYT-6091 reduced tumor perfusion by 80% of control (no treatment), 4 hours post administration. However, soluble TNF was toxic to the tumor burdened mice and resulted in 40% mortality alone and 100% mortality when combined with hyperthermia. Conversely, no toxicities were noted with CYT-6091 alone or when combined with hyperthermia. Additionally, CYT-6091 combined with heat yielded significant tumor regression in vivo as compared to heat or CYT-6091 alone as demonstrated by tumor growth delay. Pretreatment with soluble TNF or CYT-6091 followed by heating reduced in vitro tumor and endothelial cell survival by 40–50% (TNF) and 70–75% (CYT-6091) of the control cell (i.e. tumor and endothelial) values, respectively. Conclusions: CYT-6091, by selectively delivering TNF to solid tumors, improves the safety of TNF treatment. In addition, the targeted delivery of TNF augments cancer thermal therapy efficacy possibly by inducing a tumor-localized inflammatory response.

Synergistic Effects of Radiation and β-Lapachone in DU-145 Human Prostate Cancer Cells In Vitro

Abstract Suzuki, M., Amano, M., Choi, J. H., Park, H. J., Williams, B. W., Ono, K. and Song, C. W. Synergistic Effects of Radiation and β-Lapachone in DU-145 Human Prostate Cancer Cells In Vitro. Radiat. Res. 165, 525–531 (2006). It has been reported that β-lapachone (β-lap), a bioreductive anti-cancer drug, synergistically interacts with ionizing radiation and that the sensitivity of cells to β-lap is closely related to the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1). Here we report the results of our studies of mechanisms underlying the synergistic interaction of β-lap and radiation in killing cancer cells using the DU-145 human prostate cancer cell line. The clonogenic cell death caused by the combination of radiation and β-lap was synergistic when β-lap was administered 0–10 h after irradiation but not when it was given before irradiation. The expression and activity of NQO1 increased significantly and remained elevated for longer than 12 h after 4 Gy irradiation, suggesting that the long-lasting elevation of NQO1 sensitized the cells to β-lap. Studies with split-dose irradiation demonstrated that β-lap given immediately after irradiation effectively inhibited sublethal radiation damage (SLD) repair. Taken together, these results lead us to conclude that the synergistic interaction between β-lap and radiation in killing cells is the result of two distinct mechanisms: First, radiation sensitizes cells to β-lap by up-regulating NQO1, and second, β-lap sensitizes cells to radiation by inhibiting SLD repair. The combination of β-lap and radiotherapy is potentially promising modality for the treatment of cancer in humans.

Arsenic trioxide induces selective tumour vascular damage via oxidative stress and increases thermosensitivity of tumours

It has previously been found that the anti-leukaemia agent Arsenic Trioxide (ATO) causes vascular shutdown in solid tumours and markedly sensitizes tumours to hyperthermia. The present study was designed to evaluate the mechanism of action and dose-dependence of ATO-induced thermosensitization in FSaII and SCK murine tumours. The role of oxidative stress was studied by observing ATO-induced vascular shutdown in vivo and ATO-induced endothelial cell adhesion molecule expression in vitro in the presence or absence of an antioxidant. It was found that a dose as low as 2 mg/kg ATO impaired vascular function, as estimated by 86Rb uptake, in the tumour. The degree of tumour growth delay induced by 1 h of hyperthermia at 42.5°C, applied 2 h after ATO injection, was proportional to the dose of ATO administered. In addition, it was found that ATO can directly thermosensitize tumour cells in vitro. The development of massive tissue necrosis in the tumour was observed in the days after treatment, especially with the combination of ATO and heating. ATO-induced adhesion molecule expression in vitro was abolished when the anti-oxidant n-acetyl-cysteine (NAC) was introduced prior to exposure, while the addition of NAC in vivo partially blocked ATO-induced vascular shutdown. These results suggest that the expression of adhesion molecules by the vasculature due to oxidative stress contribute to the ATO-induced selective tumour vascular effects observed and that the clinical use of ATO to increase tumour thermosensitivity via direct cellular and vascular effects appears feasible.

Response of Breast Cancer Cells and Cancer Stem Cells to Metformin and Hyperthermia Alone or Combined

Metformin, the most widely prescribed drug for treatment of type 2 diabetes, has been shown to exert significant anticancer effects. Hyperthermia has been known to kill cancer cells and enhance the efficacy of various anti-cancer drugs and radiotherapy. We investigated the combined effects of metformin and hyperthermia against MCF-7 and MDA-MB-231 human breast cancer cell, and MIA PaCa-2 human pancreatic cancer cells. Incubation of breast cancer cells with 0.5–10 mM metformin for 48 h caused significant clonogenic cell death. Culturing breast cancer cells with 30 µM metformin, clinically relevant plasma concentration of metformin, significantly reduced the survival of cancer cells. Importantly, metformin was preferentially cytotoxic to CD44high/CD24low cells of MCF-7 cells and, CD44high/CD24high cells of MIA PaCa-2 cells, which are known to be cancer stem cells (CSCs) of MCF-7 cells and MIA PaCa-2 cells, respectively. Heating at 42°C for 1 h was slightly toxic to both cancer cells and CSCs, and it markedly enhanced the efficacy of metformin to kill cancer cells and CSCs. Metformin has been reported to activate AMPK, thereby suppressing mTOR, which plays an important role for protein synthesis, cell cycle progression, and cell survival. For the first time, we show that hyperthermia activates AMPK and inactivates mTOR and its downstream effector S6K. Furthermore, hyperthermia potentiated the effect of metformin to activate AMPK and inactivate mTOR and S6K. Cell proliferation was markedly suppressed by metformin or combination of metformin and hyperthermia, which could be attributed to activation of AMPK leading to inactivation of mTOR. It is conclude that the effects of metformin against cancer cells including CSCs can be markedly enhanced by hyperthermia.

Use of a Fluorescently Labeled Poly-Caspase Inhibitor for In Vivo Detection of Apoptosis Related to Vascular-Targeting Agent Arsenic Trioxide for Cancer Therapy

Arsenic trioxide (ATO, Trisenox) is a potent anti-vascular agent and significantly enhances hyperthermia and radiation response. To understand the mechanism of the anti-tumor effect in vivo we imaged the binding of a fluorescently-labeled poly-caspase inhibitor (FLIVO) in real time before and 3 h or 24 h after injection of 8 mg/kg ATO. FSaII tumors were grown in dorsal skin-fold window chambers or on the rear limb and we observed substantial polycaspase binding associated with vascular damage induced by ATO treatment at 3 and 24 h after ATO injection. Flow cytometric analysis of cells dissociated from the imaged tumor confirmed cellular uptake and binding of the FLIVO probe. Apoptosis appears to be a major mode of cell death induced by ATO in the tumor and the use of fluorescently tagged caspase inhibitors to assess cell death in live animals appears feasible to monitor and/or confirm anti-tumor effects of therapy.

Antiangiogenesis therapy using a novel angiogenesis inhibitor, anginex, following radiation causes tumor growth delay

BackgroundThe present study investigated whether treatment with anginex, a novel antiangiogenic peptide, could block re-vascularization after radiation treatment.MethodsA squamous cell (SCCVII) xenograft tumor mouse model was employed to assess the effects of anginex given post-radiation on tumor growth, microvessel density (MVD), and oxygen levels. The oxygen status was determined by the partial pressure of O2.ResultsTumors in untreated mice increased threefold in 7.0 days, anginex-treated tumors (10 mg/kg intraperitoneal, twice) required 7.3 ± 0.9 days, and tumors exposed to 8-Gy radiation increased threefold over 11 days. Combination treatment of anginex and radiation caused the tumors to grow threefold in 16.1 ± 1.6 days, a delay which was significant and deemed supra-additive. Oxygen levels in tumors treated by stand-alone or combination therapies were significantly reduced; for example from 19.5 ± 4.9 mmHg in controls to 9.7 ± 1.9 mmHg in combination-treated, size-matched tumors. In addition, immunohistochemistry showed a decrease in MVD in the tumors treated with anginex, radiation, or the combination. These results suggest that a combination of anginex and radiation can greatly affect the amount of functional vasculature in tumors and prolong radiation-induced tumor regression.ConclusionAntiangiogenesis therapy with anginex, in addition to radiotherapy, will be useful by blocking angiogenesis-dependent regrowth of vessels.

Heat-Induced Up-Regulation of NAD(P)H:Quinone Oxidoreductase Potentiates Anticancer Effects of β-Lapachone

Purpose: The purpose of the present study was to evaluate the efficacy of mild hyperthermia to potentiate the anticancer effects of β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione) by up-regulating NAD(P)H:quinone oxidoreductase (NQO1) in cancer cells. Experimental Design: Effects of β-lapachone alone or in combination with mild heating on the clonogenic survival of FSaII fibrosarcoma cells of C3H mice and A549 human lung tumor cells in vitro was determined. Effects of heating on the NQO1 level in the cancer cells in vitro were assessed using Western blot analysis for NQO1 expression, biochemical determination of NQO1 activity, and immunofluorescence microscopy for NQO1 expression. Growth of FSaII tumors in the hind legs of C3H mice was determined after treating the host mice with i.p. injection of 45 mg/kg β-lapachone followed by heating the tumors at 42°C for 1 hour every other day for four times. Results: Incubation of FSaII tumor cells and A549 tumor cells with β-lapachone at 37°C reduced clonogenic survival of the cells in dose-dependent and incubation time–dependent manner. NQO1 level in the cancer cells in vitro increased within 1 hour after heating at 42°C for 1 hour and remained elevated for >72 hours. The clonogenic cell death caused by β-lapachone increased in parallel with the increase in NQO1 levels in heated cells. Heating FSaII tumors in the legs of C3H mice enhanced the effect of i.p.-injected β-lapachone in suppressing tumor growth. Conclusion: We observed for the first time that mild heat shock up-regulates NQO1 in tumor cells. The heat-induced up-regulation of NQO1 enhanced the anticancer effects of β-lapachone in vitro and in vivo.