Preparation, characterization, in-vitro drug release and cellular uptake of poly(caprolactone) grafted dextran copolymeric nanoparticles loaded with anticancer drug.

Biodegradable and biocompatible polymers that are engineered to nanostructures play a key role in providing solution for sustained chemotherapy. This study is focused on preparation, drug encapsulation efficiency, in-vitro drug release, in-vitro cellular uptake and cell viability of poly(caprolactone) grafted dextran (PGD) nanoparticles (NPs) formulation containing vinblastine as the anticancer drug. Drug-loaded PGD NPs were prepared by a modified oil/water emulsion method and characterized by laser light scattering, atomic force microscopy (AFM), and zeta potential. The drug encapsulation efficiency was determined spectrophotometrically and in-vitro drug release was estimated using dialysis bag. Breast cancer cell line (MCF-7) was used to image and measure the cellular uptake of fluorescent PGD NPs. Cancer cell viability was assessed by treating MCF-7 cells with vinblastine-loaded PGD NPs by crystal violet staining method. Result showed that the vinblastine-loaded PGD NPs were superior in properties such as drug encapsulation efficiency, the cellular uptake and the cancer cell mortality.

GeO2 fibers: preparation, morphology and photoluminescence property.

Nanomicron to submicron fibers of GeO(2) have been prepared using poly(vinyl acetate) and germanium dioxide sol by electrospinning followed by high temperature calcination. The morphology of the fibers have been studied by scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. X-ray diffraction indicates that the fibers are single crystal with hexagonal alpha-phase quartz-like structure. At room temperature, the fibers show photoluminescence under excitation at 325 nm. The fibers may have potential applications in one-dimensional optoelectronic nanodevices.

Increased tumor cures using combined radiosurgery and BCNU in the treatment of 9l glioma in the rat brain.

PURPOSE: Radiosurgery refers to the delivery of high, single focused beams of ionizing radiation to defined intracranial lesions. 1,3 Bis[2-chloroethyl]-1-nitrosourea (BCNU) and cis-diammine-1, 1-cyclobutane-dicarboxylate platinum (II) (carboplatin) are commonly used cytotoxic agents for the treatment of malignant gliomas of the brain. Drug therapies have exhibited a modest enhanced cell killing when combined with radiation in experimental animal tumor systems. The purpose of the present study was to investigate the role of cytotoxic drugs, such as BCNU and carboplatin, in combination with a single high dose of radiosurgery on the tumor control rates of 9L tumors in the rat brain. METHODS AND MATERIALS: Combined radiosurgery (25 Gy single dose) and/or chemotherapy (a single dose of BCNU, 7 mg/kg, i.p. 1.5 or 16 h prior to or 16 h after irradiation or a single dose of carboplatin, 30 mg/kg, administered either 1 h or 4 h prior to irradiation) was delivered 12 days after stereotactic tumor implantation. For dose escalation study, 4-10 mg/kg of BCNU was used. RESULTS: The radiation alone group showed a dose-dependent survival. A single dose of 25 Gy to the control group resulted in an increase of the median survival time from 20 days to 42 days, but all animals died of the tumor in 50 days. A significant prolongation of the median survival time of animals was more than 100 days, resulting in animal cures of 50% or more when combined with radiosurgery (25 Gy) and BCNU (7 mg/kg). BCNU alone did not prolong the median survival time of the animal with the 9L brain tumor. In contrast, there was no survival improvement when the animals were treated with combined radiosurgery and carboplatin. None of the long-term surviving animals showed any significant brain tissue damage as evaluated by histopathology and clinical observations. CONCLUSION: The data clearly suggest that the combined modalities of radiosurgery and concomitant BCNU represent an effective therapeutic regimen in the treatment of radioresistant human malignant gliomas of the brain. This study represents the first experimental report of the effectiveness of combined chemotherapy and radiosurgery.

Fractionated radiosurgery for 9L gliosarcoma in the rat brain.

PURPOSE: Fractionated radiosurgery is being carried out in the clinic to improve the therapeutic ratio of single-dose radiosurgery using various fractionation schemes. Because there is a paucity of experimental radiobiological data in the literature on the tumor response and late-responding normal tissue of critical intracranial structures to radiosurgery, the present animal study was designed to compare the response following a single high dose of radiation with that obtained from calculated fractionated doses of radiosurgery. METHODS AND MATERIALS: Male Fischer rats with 9L gliosarcoma growing in their brains were stereotactically irradiated and assayed for the tumor control rate and brain tissue damage. The radiation dose needed for 50% tumor control (TCD50) was used as the endpoint of the efficacy of radiosurgery. Normal brain damage was measured histologically following a period of time over 270 days. Histological evaluation included hematoxylin-eosin (H & E), Luxol fast blue and periodic acid Schiff (LFB/PAS) for the presence of myelin and glial fibrillary acidic protein (GFAP) for the assessment of astrocytic re-activity. The optical density of optic nerves and chiasms staining with LFB/PAS was quantitatively measured using a computer image analysis to assess the magnitude of demyelination. RESULTS: Radiosurgery (RS) was found to be more effective in curing small tumors than large tumors. The dose required to control 50% of the tumored animals for 120 days was 24, 31, and 40 Gy for 2-, 6-, and 12-day-old tumors, respectively. Using 12-day-old brain tumors, two fractions of 23.5 Gy and three fractions of 18.5 Gy were found to be equivalent to the single dose of 35 Gy for tumor control. For normal brain damages, the visual pathways including optic nerves and chiasm were found to be highly radiosensitive structures. A single dose of 35 Gy produced 100% severe optic neuropathy. The fractionated RS regimens spared substantial optic nerve damage. CONCLUSION: The present data provide a strong radiobiological rationale for the use of fractionated RS in the treatment of tumors located near critical normal structures, including visual pathways. The sparing effect of fractionated RS is greater for late-responding tissues, relative to the rapidly proliferating tumor tissues. This report also characterizes the dose/time tolerance relationship of optic neuropathy after single and fractionated RS.

Selective radiosensitization of 9L glioma in the brain transduced with double suicide fusion gene.

PURPOSE: Suicide gene therapy has proved to be successful in enhancing the therapeutic index by sensitizing genetically modified tumor cells to prodrugs. Two of the most widely studied suicide genes, herpes simplex virus type 1 thymidine kinase and Escherichia coli cytosine deaminase, have proved effective at selectively eliminating malignant tumor cells. We previously demonstrated that transduced 9L glioma cells expressing E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase concomitantly as a fusion protein exhibited greater levels of targeted cytotoxicity and radiosensitization than could be achieved by single suicide gene therapy. The present in vivo studies were carried out to determine whether double suicide gene therapy would enhance the tumor control rate of orthotopically implanted malignant glioma growing in the brain when coupled with radiotherapy. MATERIALS AND METHODS: Rat 9L gliosarcoma cells were transfected with retroviral vectors containing an E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase fusion gene and maintained in Dulbecco's modified Eagle's medium. The antitumor response of 9L E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase tumors growing in the brain of Fischer rats was evaluated with small tumors (6-day-old tumors) versus large tumors (14-day-old tumors) against single versus double prodrug treatments. In the large brain tumors, the therapeutic efficacy of the combined single and double prodrugs coupled with radiotherapy was evaluated. RESULTS: Double suicide gene therapy using two prodrugs, 5-fluorocytosine (500 mg/kg) and ganciclovir (30 mg/kg), was effective in achieving long-term tumor control (50% survival) against early-stage brain tumors (6 days after implantation) but was only marginally effective against advanced stage tumors (14 days old). However, when these prodrugs were combined with radiotherapy and double suicide gene therapy against advanced-stage tumors, more than 70% of the animals were cured, whereas radiotherapy alone (20 Gy) failed to achieve any cure at all. Combined radiotherapy and single prodrug therapy showed a moderate increase in the animal survival rate (17% and 40% for 5-fluorocytosine and ganciclovir, respectively) but was inferior to the combination therapy of radiation and double prodrugs. CONCLUSION: The present in vivo results indicate that double suicide gene therapy combined with radiotherapy may represent a new, effective approach to achieve a high tumor cure rate without producing any excessive normal tissue damage.

Tumor control of locally advanced prostate cancer following combined estramustine, vinblastine, and radiation therapy.

PURPOSE: A prospective phase II study was carried out to determine whether estramustine phosphate (EMP) plus vinblastine (VBL) in combination with radiotherapy (RT) would improve the control of locally advanced prostate cancer. The rationale for combining EMP plus VBL with RT was based on the clinical and radiobiological data that EMP plus VBL acted as an excellent radiation sensitizer in cultured human prostatic carcinoma cells with the property of tissue selectivity. The combined EMP and VBL were well tolerated in the phase II clinical study of patients with advanced prostate cancer. MATERIALS AND METHODS: Between January 1991 and July 1996, 65 patients, stage T2 (B2) through stage T4 (D1), were entered into the study. Gleason pattern scores ranged from 4 to 10. Pretreatment prostate-specific antigen (PSA) was as follows: < 20 in 21 patients (32%), 20 to 50 in 23 patients (35%), and > 50 in 21 patients (32%). The median age was 70 years (55-83). All patients were treated with megavoltage beam radiation with a total tumor dose of 65 to 70 Gy. Oral EMP 450 mg/m2 daily and VBL 3 mg/m2 weekly were given concomitantly in 46 patients during the 7- to 7 1/2-week course of radiotherapy. RESULTS: All patients showed prompt and complete tumor regression on digital rectal examination at 6 weeks following the completion of treatment. Median follow-up time is 43 months (3-65). PSA fell to an undetectable level by 6 weeks in 56 of 65 patients (86%). For the whole group at 5 years clinical control was 81%, but biochemical control (PSA < 4 ng/mL) was 48%. The likelihood of being free of biochemical relapse at 5 years was a function of initial PSA value (PSA < 20 in 64% of the cases, 21-50 in 60%, and > 50 in 0%). The biochemical-relapse-free survival at 5 years for each stage was T2, 49%; T3, 38%; and T4, 17%. In particular, a group of patients with pretreatment PSA levels of 20 to 50 ng/mL responded quite favorably to the present combined regimen in that only 40% of the patients showed a biochemical failure at 5 years, considering the high level of initial PSA. CONCLUSIONS: The present combined approach is effective in achieving a high rate of tumor control with no disproportionately enhanced side effects. The rapid regression of the tumor nodules and sustained freedom from biochemical relapse suggest excellent long-term tumor control, especially in the group of patients with pretreatment PSA levels of 20 to 50 ng/mL.

Ethacrynic acid: a novel radiation enhancer in human carcinoma cells.

PURPOSE: Because agents that interfere with thiol metabolism and glutathione S-transferase (GST) functions have been shown to enhance antitumor effects of alkylating agents in vitro and in vivo, the present study was conceived on the basis that an inhibitor of GST would enhance the radiation response of some selected human carcinoma cells. Ethacrynic acid (EA) was chosen for the study because it is an effective inhibitor of GST and is a well known diuretic in humans. METHODS AND MATERIALS: Experiments were carried out with well-established human tumor cells in culture growing in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS). Cell lines used were MCF-7, MCF-7 adriamycin resistant (AR) cells (breast carcinoma), HT-29 cells (colon carcinoma), DU-145 cells (prostate carcinoma), and U-373 cells (malignant glioma). Cell survival following the exposure of cells to drug alone, radiation alone, and a combined treatment was assayed by determining the colony-forming ability of single plated cells in culture to obtain dose-survival curves. The drug enhancement ratio was correlated with levels of GST. RESULTS: The cytotoxicity of EA was most pronounced in MCF-7, U-373, and DU-145 cells compared to MCF-7 AR and HT-29 cells. The levels of GST activity were found to be lower in those EA-sensitive cells. A significant radiation enhancement was obtained with EA-sensitive cells exposed to nontoxic concentrations of the drug immediately before or after irradiation. The sensitizer enhancement ratio (SER) of MCF-7 cells was 1.55 with EA (20 micrograms/ml), while the SER of MCF-7 AR was less than 1.1. Based on five different human tumor cells, a clear inverse relationship was demonstrated between the magnitude of SER and GST levels of tumor cells prior to the combined treatment. CONCLUSION: The present results suggest that EA, which acts as both a reversible and irreversible inhibitor of GST activity, could significantly enhance the radiation response of human cancer cells and the level of GST in tumor cells may predict the magnitude of radiation enhancement with EA. Ethacrynic acid would be an excellent drug as a radiosensitizer for further in vivo tumor study.

Preferential radiosensitization of human prostatic carcinoma cells by mild hyperthermia.

PURPOSE: Recent cell culture studies by us and others suggest that some human carcinoma cells are more sensitive to heat than are rodent cells following mild hyperthermia. In studying the cellular mechanism of enhanced thermosensitivity of human tumor cells to hyperthermia, prostatic carcinoma cells of human origin were found to be more sensitive to mild hyperthermia than other human cancer cells. The present study was designed to determine the magnitude of radiosensitization of human prostatic carcinoma cells by mild hyperthermia and to examine whether the thermal radiosensitization is related to the intrinsic thermosensitivity of cancer cells. METHODS AND MATERIALS: Two human prostatic carcinoma cell lines (DU-145 and PC-3) and other carcinoma cells of human origin, in particular, colon (HT-29), breast (MCF-7), lung (A-549), and brain (U-251) were exposed to temperatures of 40-41 degrees C. Single acute dose rate radiation and fractionated radiation were combined with mild hyperthermia to determine thermal radiosensitization. The end point of the study was the colony-forming ability of single-plated cells. RESULTS: DU-145 and PC-3 cells were found to be exceedingly thermosensitive to 41 degrees C for 24 h, relative to other cancer cell lines. Ninety percent of the prostatic cancer cells were killed by a 24 h heat exposure. Prostatic carcinoma cells exposed to a short duration of heating at 41 degrees C for 2 h resulted in a substantial enhancement of radiation-induced cytotoxicity. The thermal enhancement ratios (TERs) of single acute dose radiation following heat treatment 41 C for 2 h were 2.0 in DU-145 cells and 1.4 in PC-3 cells. The TERs of fractionated irradiation combined with continuous heating at 40 degrees C were similarly in the range of 2.1 to 1.4 in prostate carcinoma cells. No significant radiosensitization was observed in MCF-7 and HT-29 cells under the same conditions. CONCLUSION: The present data suggest that a significant radiosensitization of prostatic cancer cells could be obtained by the combined treatment of radiation and mild hyperthermia. Future clinical trials should be aimed at achieving mild heating and fractionated radiation therapy.

Radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells in culture transduced with cytosine deaminase gene.

Recently, use of the suicide gene, cytosine deaminase (CD), has shown a selective antitumor activity of 5-fluorocytosine (5-FC) on human colorectal carcinoma cells grown in vitro and in vivo. We hypothesized that the radiosensitivity of human colorectal carcinoma cells transduced with a retroviral vector encoding the bacterial CD gene would be selectively enhanced by the nontoxic prodrug 5-FC. The radiobiological rationale of using suicide gene therapy is based on the fact that a toxic metabolite of 5-FC, 5-fluorouracil, is a well-known radiation enhancer for the treatment of gastrointestinal and other tumors. 5-FC was found to enhance selectively the radiation cytotoxicity of human colorectal carcinoma cells expressing the CD gene. Colorectal carcinoma cells transduced with the CD gene (WiDr-CD) were highly sensitive to radiation compared with parental cells (WiDr) when exposed to 20 microgram/ml 5-FC for 72 h prior to irradiation. The sensitization enhancement ratio was 2.38. This magnitude of radiation enhancement is comparable to that obtained with 5-fluorouracil. These results suggest that the addition of radiation would substantially improve the therapeutic potential of CD gene therapy for the treatment of locally advanced colorectal carcinomas.

Estramustine: a novel radiation enhancer in human carcinoma cells.

PURPOSE: Estramustine (EM), an antimicrotubule agent, binds microtubule-associated proteins, causes spindle disassembly, and arrests cells at the late G2/M phase of the cell cycle. Since cells in the G2/M phase are the most radiosensitive and some human cancer cells contain high level of EM-binding protein, experiments were carried out to determine whether radiation sensitization could be obtained in human carcinoma cells. METHODS AND MATERIALS: Cells containing a high level of EM-binding protein such as prostate carcinoma (DU-145), breast carcinoma (MCF-7), and malignant glioma (U-251) were used to demonstrate radiosensitization. Cervical carcinoma (HeLa-S3) and colon carcinoma (HT-29) cells which are not known to contain EM-binding protein were also employed. Cell survival was assayed by the colony forming ability of single plated cells in culture to obtain dose-survival curves. RESULTS: Pretreatment of DU-145, MCF-7, and U-251 cells to a nontoxic concentration (5 microM) of EM for more than one cell cycle time, substantially enhanced the radiation-induced cytotoxicity. The sensitizer enhancement ratio of these cells ranged from 1.35-1.52. The magnitude of the enhancement was dependent on the drug concentration and exposure time. The rate of cell accumulation in G2/M phase, as determined by flow cytometry, increased with longer treatment time in the cell lines which showed radiosensitization. Other antimicrotubule agents such as taxol and vinblastine caused minimal or no radiosensitization at nontoxic concentrations. CONCLUSION: The data provide a radiobiological basis for using EM as a novel radiation enhancer, with the property of tissue selectivity.

Clinical and biological studies of estramustine phosphate as a novel radiation sensitizer.

PURPOSE: Estramustine phosphate (EMP), a nor-nitrogen mustard carbamate derivative of estradiol-17 beta-phosphate, causes G2/M phase arrest in treated cells through its specific binding to microtubule associated proteins. Since cells in the G2/M phase are the most radiosensitive, cell culture experiments were performed to determine whether EMP would enhance the radiosensitivity of related human tumor cells. Based on the cell culture findings and well known pharmacokinetic data in humans, a Phase II prospective study of concomitant radiotherapy (RT) and EMP plus Velban for locally advanced carcinoma of the prostate was carried out. METHODS AND MATERIALS: Three established human tumor cells, DU-145 cells (prostate), MCF-7 cells (breast), and U-251 cells (malignant glioma), were used to determine cell survival curves with and without the drug. Flow cytometry was used to obtain the cell cycle distribution of cells that were exposed to the drug for periods of 1 day to 1 week. Patients with locally advanced prostate cancer (Stages B2, C, D1) were entered into the Phase II study. All patients received a total tumor dose of 65-70 Gy over 7 weeks. Oral EMP was administered daily and Velban was administered weekly, concomitantly during the course of RT. RESULTS: Radiosensitization was dependent on the exposure time and the drug concentration prior to radiation. No radiosensitization was obtained when cells were exposed to the drug after irradiation. The enhancement ratios varied from 1.3-1.6 at the 10% survival level. All patients who received the combined RT and EMP plus Velban achieved complete response (n = 27). The rate of PSA (prostate specific antigen) reduction was very prompt compared to that of the RT alone group. There was not disproportionately enhanced side effects for the combined regimen. CONCLUSION: EMP enhances radiation induced cytotoxicity in several human tumor cells in culture. The effect is most significant after prolonged exposure to the drug before irradiation. Documented G2/M phase cell cycle block by EMP is the likely mechanism of radiosensitization. The preliminary clinical findings with the combined RT and drugs are highly encouraging.

Enhancement of radiation response on human carcinoma cells in culture by pentoxifylline.

PURPOSE: Pentoxifylline, a methylxanthine, has been shown to improve tumor tissue oxygenation in hypoxic murine tumors and prevent the late radiation injury of normal tissues in mice. The present cell culture studies were carried out to determine whether pentoxifylline would enhance the cellular radiation response of several human carcinoma cells in culture under various culture conditions. METHODS AND MATERIALS: Experiments were carried out with three human carcinoma cells grown in Eagle's MEM supplemented with 10% FCS. Cell survival was assayed by the colony forming ability of single plated cells to obtain dose-survival curves. RESULTS: Cells irradiated and exposed to pentoxifylline for 24 hr showed a significant enhancement of radiation-induced cytotoxicity. Pre-irradiation treatment with the drug did not change cellular response to radiation. The magnitude of radiation enhancement was dependent on the concentration of drug and exposure time up to the time corresponding to one cell cycle time. Among the three human carcinoma cells used (HeLa S-3 cervix carcinoma, MCF-7 breast carcinoma, and HT-29 colon carcinoma), HeLa S-3 cells were most susceptible to the combined treatment with the enhancement ratio of 1.4 at 1 mM. Among the derivatives of methylxanthines, caffeine and pentoxifylline were equally effective on a molar basis. The combined effect of pentoxifylline and purine nucleosides, including guanosine and deoxyguanosine, further enhanced the sensitizing effect of pentoxifylline. CONCLUSION: The present data along with other radiobiological effects of the drug suggest that pentoxifylline should be considered as a radiation enhancer for clinical radiotherapy.

Therapeutic options for localized carcinoma of the prostate: the role of external beam radiation therapy.

During the last three decades the use of ionizing radiation, both external beam radiotherapy and interstitial radionuclide implant (brachytherapy), has greatly increased for the treatment of cancer of the prostate. The increased use of radiation therapy is in part due to the technological advance of high-energy megavoltage units as well as the steadily improving long-term results of radiotherapy. Because of the comparable tumor control rates, patients with early stage prostate cancer have several therapeutic options for curative treatment including radical prostatectomy, external beam radiation therapy, and interstitial brachytherapy. The role of external beam radiotherapy is discussed in terms of the primary management of early and locally advanced prostate cancers as well as the treatment of residual or recurrent disease after prostatectomy. A new approach combines antimitotic chemotherapeutic agents and radiation therapy for the treatment of locally advanced cancers of the prostate.

Potentiation of radiation response in human carcinoma cells in vitro and murine fibrosarcoma in vivo by topotecan, an inhibitor of DNA topoisomerase I.

DNA topoisomerase I, a nuclear enzyme important for solving topologic problems arising during DNA replication, has been identified as a principal target of a plant alkaloid, 20(s)-camptothecin. In view of the profound biochemical effects of camptothecin and its analogues on DNA replication and the differential cytotoxic effects on human tumors in xenografts, experiments were performed to determine whether topotecan, a camptothecin analogue, would potentiate the radiation effects on human carcinoma cells in culture and murine fibrosarcoma in mice. Cell culture studies showed that a dose dependent reduction in cell survival was obtained with a 4 hr exposure of the drug following irradiation of cells. No enhancement of cell killing was seen when cells were treated with the drug before irradiation. Preliminary in vivo tumor studies showed a significant radiosensitizing effect of topotecan that was both drug dose (20 mg/kg) and time sequence (4 hr before irradiation) dependent. There was no enhanced skin reaction following the combined treatments.