Pawel Zwolak

Biology of Bone Cancer Pain

Bone cancer pain is a devastating manifestation of metastatic cancer. Unfortunately, current therapies can be ineffective, and when they are effective, the duration of the patients survival typically exceeds the duration of pain relief. New, mechanistically based therapies are desperately needed. Study of experimental animal models has provided insight into the mechanisms that drive bone cancer pain and provides an opportunity for developing targeted therapies. Mechanisms that drive bone cancer pain include tumor-directed osteoclast-mediated osteolysis, tumor cells themselves, tumor-induced nerve injury, stimulation of transient receptor potential vanilloid type 1 ion channel, endothelin A, and host cell production of nerve growth factor. Current and future therapies include external beam radiation, osteoclast-targeted inhibiting agents, anti-inflammatory drugs, transient receptor potential vanilloid type 1 antagonists, and antibody therapies that target nerve growth factor or tumor angiogenesis. It is likely that a combination of these therapies will be superior to any one therapy alone.

Advances in understanding bone cancer pain

Experimental animal models of bone cancer pain have emerged and findings have provided a unique glimpse into unraveling the mechanism that drives this debilitating condition. Key contributors to the generation and maintenance of bone cancer pain are tumor‐induced osteolysis, tumor itself, and production of nociceptive mediators in the bone‐tumor microenvironment. J. Cell. Biochem.

Cytotoxic effect of zoledronic acid-loaded bone cement on giant cell tumor, multiple myeloma, and renal cell carcinoma cell lines.

BACKGROUND Local recurrence with subsequent osteolysis is a problem after intralesional curettage of giant cell tumor of bone, myeloma, and metastatic carcinoma. The bisphosphonate zoledronic acid (zoledronate) has been shown to reduce osteoclast activity, and its local administration is a potentially attractive therapy, especially for the osteoclast-rich giant cell tumor. The aim of this study was to analyze the elution dynamics of zoledronic acid release from acrylic bone cement and its in vitro antitumor efficacy. METHODS Various concentrations of zoledronic acid were mixed with bone cement and placed in distilled water. The concentration in the water was measured daily for fourteen days. The cytotoxic effects of the dissolved zoledronic acid on cultures of multiple myeloma, giant cell tumor, and renal cell carcinoma cells were tested with use of the MTT assay (tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye) and analyzed according to the zoledronic acid concentration and the elapsed time. RESULTS The release of zoledronic acid was greatest during the first twenty-four hours for all concentrations and decreased rapidly during the next forty-eight hours to reach a plateau after four days. The proliferation assay (MTT) showed zoledronic acid to have significant cytotoxicity in cultures of stromal giant cell tumor, multiple myeloma, and renal cell carcinoma cells. In addition, zoledronic acid decreased the number of viable tumor cells in a dose-dependent manner. Renal cell carcinoma from bone (RBM1-IT4) and stromal giant cell tumor of bone were more susceptible to zoledronic acid than was multiple myeloma. CONCLUSIONS The method presented in our study is a reproducible technique for evaluating zoledronic acid elution from bone cement and determining its impact on tumor growth. Zoledronic acid is released from bone cement, remains biologically active despite the polymerization of cement, and inhibits the in vitro growth of cell lines from giant cell tumor of bone, myeloma, and renal cell carcinoma.

Addition of receptor tyrosine kinase inhibitor to radiation increases tumour control in an orthotopic murine model of breast cancer metastasis in bone

The receptor tyrosine kinase inhibitor, SU11248, was added to localised radiation to evaluate the response of bone metastases and to define the basic mechanism of radiosensitisation. Treatment with SU11248 and radiation was assessed in vitro using cultured 4T1 breast cancer cells and in vivo using an orthotopic 4T1 murine mammary tumour model of breast cancer bone metastasis. Cultured 4T1 cells treated with SU11248 (1 microM) and radiation (10 Gy) showed an almost 7.5-fold increase in caspase-mediated apoptosis after 24 h of incubation, compared to either treatment alone. Mice treated with SU11248 (40 mg/kg/daily) and radiation (15 Gy/single-dose) had a relatively greater reduction in tumour growth, bone osteolysis, osteoclast maturation and microvessel density. Combined modality treatment resulted in improvements in behavioural pain assessment scores and normalisation of neurochemical changes in the spinal cord receiving primary afferent innervation from tumour-bearing femora. Our study demonstrates that SU11248 enhances the radiation control of metastatic breast tumours in bone and tumour-induced pain.

Protein kinase C-β inhibitor enzastaurin (LY317615.HCI) enhances radiation control of murine breast cancer in an orthotopic model of bone metastasis

SummaryRadiation therapy is a widely used treatment for metastatic bone cancer, but the rapid onset of tumor radioresistance is a major problem. We investigated the radiosensitizing effect of enzastaurin, a protein kinase Cβ (PKCβ) inhibitor, on bone tumor growth and tumor-related pain. We found that enzastaurin enhanced the effect of ionizing radiation on cultured murine 4T1 breast cancer and murine endothelial cells, suppressing their proliferation and colony formation. Enzastaurin and ionizing radiation also induced caspase-mediated apoptosis of 4T1 cells to a greater degree than radiation alone. Enzastaurin treatment of 4T1 cells blocked the phosphorylation of PKCβ, as well as Ras and two of its downstream effectors ERK1/2 and RAL-GTP. Using an orthotopic model of bone metastasis, we observed that a combination of enzastaurin and localized radiation treatment reduced tumor blood vessel density, bone destruction and pain compared to single modality treatment. In conclusion, we demonstrate that inhibition of PKCβ in combination with localized radiation treatment suppresses tumor growth and alleviates pain as compared to radiation-only treatment. We also show that the radiosensitizing effect of enzastaurin is associated with suppression of tumor cell proliferation and tumor-induced angiogenesis possibly through inhibition of the Ras pathway.

Novel Ras pathway inhibitor induces apoptosis and growth inhibition of K-ras-mutated cancer cells in vitro and in vivo

MT477 is a novel quinoline with potential activity in Ras-mutated cancers. In this study, MT477 preferentially inhibited the proliferation of K-ras-mutated human pulmonary (A549) and pancreatic (MiaPaCa-2) adenocarcinoma cell lines, compared with a non-Ras-mutated human lung squamous carcinoma cell line (H226) and normal human lung fibroblasts. MT477 treatment induced apoptosis in A549 cells and was associated with caspase-3 activation. MT477 also induced sub-G1 cell-cycle arrest in A549 cells. Although we found that MT477 partially inhibited protein kinase C (PKC), it inhibited Ras directly followed in time by inhibition of 2 Ras downstream molecules, Erk1/2 and Ral. MT477 also caused a reorganization of the actin cytoskeleton and formation of filopodias in A549 cells; this event may lead to decreased migration and invasion of tumor cells. In a xenograft mouse model, A549 tumor growth was inhibited significantly by MT477 at a dose of 1 mg/kg (P < 0.05 vs vehicle control). Taken together, these results support the conclusion that MT477 acts as a direct Ras inhibitor. This quinoline, therefore, could potentially be active in Ras-mutated cancers and could be developed extensively as an anticancer molecule with this in mind.

Local irradiation in combination with bevacizumab enhances radiation control of bone destruction and cancer-induced pain in a model of bone metastases

Skeletal metastases are a major source of morbidity for cancer patients. The purpose of this study was to evaluate the effects of megavoltage irradiation and antiangiogenic therapy on metastatic bone cancer. A tumor xenograft model was prepared in C3H/Scid mice using 4T1 murine breast carcinoma cells. Twenty‐eight mice bearing tumors were treated with either bevacizumab (15 mg/kg), local megavoltage irradiation (30 Gy in 1 fraction), combination of bevacizumab and local megavoltage irradiation or physiologic saline solution (control group). Tumor area, bone destruction, tumor microvessel density, pain‐associated behaviors and expression of substance P were assessed. Combined modality treatment reduced the frequency of pain‐associated behaviors, decreased levels of nociceptive protein expression in the spinal cord, maintained cortical integrity and decreased the density of microvessels as compared to single modality treatments. We conclude that concurrent antiangiogenic therapy and localized radiotherapy for the treatment of bone metastases warrants further evaluation in human clinical trials.

Novel Cytosine Deaminase Fusion Gene Enhances the Effect of Radiation on Breast Cancer in Bone by Reducing Tumor Burden, Osteolysis, and Skeletal Fracture

Background: Painful breast carcinoma metastases in bone are a common manifestation of malignant disease. Eradication of these tumors can be evasive, and as a result, skeletal morbidity increases with disease progression. Experimental Design: The treatment potential of cytosine deaminase (CD) gene therapy combined with radiation treatment was evaluated in vitro and in vivo using a 4T1 murine breast carcinoma model. 4T1 carcinoma cells were transduced with a fusion gene encoding the extracellular and transmembrane domains of the human nerve growth factor receptor and the cytoplasmic portion of the yeast CD gene (NGFR-CDy). Results and Conclusions: CD-expressing tumor cells (4TCDy) were highly sensitive to treatment by 5-fluorocytosine prodrug (P < 0.0001). 5-Fluorocytosine treatment of 4TCDy, but not 4T1 cells, enhanced the effects of radiation in vitro (P < 0.0001). 5-Fluorocytosine prodrug treatment also increased the therapeutic potential of radiation in vivo. Mice with 4TCDy intrafemoral tumors showed increased effectiveness of radiation based on improved reductions in tumor size, reductions in tumorigenic osteolysis, and a decrease in skeletal fractures (P < 0.01).

Enzastaurin renders MCF-7 breast cancer cells sensitive to radiation through reversal of radiation-induced activation of protein kinase C

Enzastaurin (LY317615.HCI), a protein kinase C (PKC)-beta inhibitor, has a radiosensitising effect on 4T1 murine breast cancer and human glioma cells; however, the exact mechanism of this action has not been evaluated. The present study investigated the effects of enzastaurin and gamma irradiation on PKC activity in MCF-7 human breast cancer cells in vitro and in vivo. Enzastaurin (5 microM) in combination with irradiation (2-8 Gy) produced a synergistic decline in MCF-7 clonogenic cell survival. Analysis of MCF-7 cells stained with Annexin V and 7-aminoactinomycin D showed a dose-dependent increase in apoptosis in response to enzastaurin (3, 5 and 7 microM) and irradiation (10 Gy) compared to irradiation alone. This pro-apoptotic effect was confirmed by increases in caspase-3 and -9 activity. In a MCF-7 xenograft model, irradiation with 25 Gy increased PKC-alpha activity by 2.5-fold compared to untreated controls, whereas PKC-epsilon and -betaII activity was increased by 1.8-fold. Radiation-induced activation of all three anti-apoptotic isoforms of PKC was reversed by pre-treatment with enzastaurin (75 mg/kg, twice daily for 3 days). We conclude that enzastaurin has a radiosensitising effect on MCF-7 human xenograft tumours through the reversal of anti-apoptotic activation of PKC isoforms.