Technical Note: Dosimetric characteristics of the ocular beam line and commissioning data for an ocular proton therapy planning system at the Proton Therapy Center Houston.

PURPOSE: To systematically analyze and present the properties of a small-field, double-scattering proton beam line intended to be used for the treatment of ocular cancer, and to provide configuration data for commission of the Eclipse Ocular Proton Planning System. METHODS: Measurements were made using ionization chambers, diodes, and films to determine dose profiles and output factors of the proton beams of the beam line at the Proton Therapy Center Houston. In parallel, Monte Carlo simulations were performed to validate the measured data and to provide additional insight into detailed beam parameters that are hard to measure, such as field size factors and a comparison of output factors as a function of circular and rectangular fields. RESULTS: The presented data comprise depth dose profiles, including distal and proximal profiles used to configure the Eclipse Ocular Proton Planning system, distal fall-off widths, lateral profiles and penumbrae sizes, as well as output factors as a function of field size, SOBP width, range shifter thickness, snout position, and source-to-surface distance. CONCLUSIONS: We have completed a comprehensive characterization of the beam line. The data will be useful to characterize proton beams in clinical and experimental small-field applications.

Intensity-Modulated Proton Therapy Adaptive Planning for Patients with Oropharyngeal Cancer.

PURPOSE: The authors aimed to illustrate the potential dose differences to clinical target volumes (CTVs) and organs-at-risk (OARs) volumes after proton adaptive treatment planning was used. PATIENTS AND METHODS: The records of 10 patients with oropharyngeal cancer were retrospectively reviewed. Each patient's treatment plan was generated by using the Eclipse treatment planning system. Verification computed tomography (CT) scan was performed during the fourth week of treatment. Deformable image registrations were performed between the 2 CT image sets, and the CTVs and major OARs were transferred to the verification CT images to generate the adaptive plan. We compared the accumulated doses to CTVs and OARs between the original and adaptive plans, as well as between the adaptive and verification plans to simulate doses that would have been delivered if the adaptive plans were not used. RESULTS: Body contours were different on planning and week-4 verification CTs. Mean volumes of all CTVs were reduced by 4% to 8% (P ≤ .04), and the volumes of left and right parotid glands also decreased (by 11% to 12%, P ≤ .004). Brainstem and oral cavity volumes did not significantly differ (all P ≥ .14). All mean doses to the CTV were decreased for up to 7% (P ≤ .04), whereas mean doses to the right parotid and oral cavity increased from a range of 5% to 8% (P ≤ .03), respectively. CONCLUSION: Verification and adaptive planning should be recommended during the course of proton therapy for patients with head and neck cancer to ensure adequate dose deliveries to the planned CTVs, while safe doses to OARs can be respected.

Quantitative analysis of treatment process time and throughput capacity for spot scanning proton therapy.

PURPOSE: To determine the patient throughput and the overall efficiency of the spot scanning system by analyzing treatment time, equipment availability, and maximum daily capacity for the current spot scanning port at Proton Therapy Center Houston and to assess the daily throughput capacity for a hypothetical spot scanning proton therapy center. METHODS: At their proton therapy center, the authors have been recording in an electronic medical record system all treatment data, including disease site, number of fields, number of fractions, delivered dose, energy, range, number of spots, and number of layers for every treatment field. The authors analyzed delivery system downtimes that had been recorded for every equipment failure and associated incidents. These data were used to evaluate the patient census, patient distribution as a function of the number of fields and total target volume, and equipment clinical availability. The duration of each treatment session from patient walk-in to patient walk-out of the spot scanning treatment room was measured for 64 patients with head and neck, central nervous system, thoracic, and genitourinary cancers. The authors retrieved data for total target volume and the numbers of layers and spots for all fields from treatment plans for a total of 271 patients (including the above 64 patients). A sensitivity analysis of daily throughput capacity was performed by varying seven parameters in a throughput capacity model. RESULTS: The mean monthly equipment clinical availability for the spot scanning port in April 2012-March 2015 was 98.5%. Approximately 1500 patients had received spot scanning proton therapy as of March 2015. The major disease sites treated in September 2012-August 2014 were the genitourinary system (34%), head and neck (30%), central nervous system (21%), and thorax (14%), with other sites accounting for the remaining 1%. Spot scanning beam delivery time increased with total target volume and accounted for approximately 30%-40% of total treatment time for the total target volumes exceeding 200 cm(3), which was the case for more than 80% of the patients in this study. When total treatment time was modeled as a function of the number of fields and total target volume, the model overestimated total treatment time by 12% on average, with a standard deviation of 32%. A sensitivity analysis of throughput capacity for a hypothetical four-room spot scanning proton therapy center identified several priority items for improvements in throughput capacity, including operation time, beam delivery time, and patient immobilization and setup time. CONCLUSIONS: The spot scanning port at our proton therapy center has operated at a high performance level and has been used to treat a large number of complex cases. Further improvements in efficiency may be feasible in the areas of facility operation, beam delivery, patient immobilization and setup, and optimization of treatment scheduling.

Proton energy optimization and reduction for intensity-modulated proton therapy.

Intensity-modulated proton therapy (IMPT) is commonly delivered via the spot-scanning technique. To 'scan' the target volume, the proton beam is controlled by varying its energy to penetrate the patient's body at different depths. Although scanning the proton beamlets or spots with the same energy can be as fast as 10-20 m s(-1), changing from one proton energy to another requires approximately two additional seconds. The total IMPT delivery time thus depends mainly on the number of proton energies used in a treatment. Current treatment planning systems typically use all proton energies that are required for the proton beam to penetrate in a range from the distal edge to the proximal edge of the target. The optimal selection of proton energies has not been well studied. In this study, we sought to determine the feasibility of optimizing and reducing the number of proton energies in IMPT planning. We proposed an iterative mixed-integer programming optimization method to select a subset of all available proton energies while satisfying dosimetric criteria. We applied our proposed method to six patient datasets: four cases of prostate cancer, one case of lung cancer, and one case of mesothelioma. The numbers of energies were reduced by 14.3%-18.9% for the prostate cancer cases, 11.0% for the lung cancer cases and 26.5% for the mesothelioma case. The results indicate that the number of proton energies used in conventionally designed IMPT plans can be reduced without degrading dosimetric performance. The IMPT delivery efficiency could be improved by energy layer optimization leading to increased throughput for a busy proton center in which a delivery system with slow energy switch is employed.

Improving spot-scanning proton therapy patient specific quality assurance with HPlusQA, a second-check dose calculation engine.

PURPOSE: The purpose of this study was to validate the use of HPlusQA, spot-scanning proton therapy (SSPT) dose calculation software developed at The University of Texas MD Anderson Cancer Center, as second-check dose calculation software for patient-specific quality assurance (PSQA). The authors also showed how HPlusQA can be used within the current PSQA framework. METHODS: The authors compared the dose calculations of HPlusQA and the Eclipse treatment planning system with 106 planar dose measurements made as part of PSQA. To determine the relative performance and the degree of correlation between HPlusQA and Eclipse, the authors compared calculated with measured point doses. Then, to determine how well HPlusQA can predict when the comparisons between Eclipse calculations and the measured dose will exceed tolerance levels, the authors compared gamma index scores for HPlusQA versus Eclipse with those of measured doses versus Eclipse. The authors introduce the αßγ transformation as a way to more easily compare gamma scores. RESULTS: The authors compared measured and calculated dose planes using the relative depth, z∕R × 100%, where z is the depth of the measurement and R is the proton beam range. For relative depths than less than 80%, both Eclipse and HPlusQA calculations were within 2 cGy of dose measurements on average. When the relative depth was greater than 80%, the agreement between the calculations and measurements fell to 4 cGy. For relative depths less than 10%, the Eclipse and HPlusQA dose discrepancies showed a negative correlation, -0.21. Otherwise, the correlation between the dose discrepancies was positive and as large as 0.6. For the dose planes in this study, HPlusQA correctly predicted when Eclipse had and had not calculated the dose to within tolerance 92% and 79% of the time, respectively. In 4 of 106 cases, HPlusQA failed to predict when the comparison between measurement and Eclipse's calculation had exceeded the tolerance levels of 3% for dose and 3 mm for distance-to-agreement. CONCLUSIONS: The authors found HPlusQA to be reasonably effective (79% ± 10%) in determining when the comparison between measured dose planes and the dose planes calculated by the Eclipse treatment planning system had exceeded the acceptable tolerance levels. When used as described in this study, HPlusQA can reduce the need for patient specific quality assurance measurements by 64%. The authors believe that the use of HPlusQA as a dose calculation second check can increase the efficiency and effectiveness of the QA process.

Use of treatment log files in spot scanning proton therapy as part of patient-specific quality assurance.

PURPOSE: The purpose of this work was to assess the monitor unit (MU) values and position accuracy of spot scanning proton beams as recorded by the daily treatment logs of the treatment control system, and furthermore establish the feasibility of using the delivered spot positions and MU values to calculate and evaluate delivered doses to patients. METHODS: To validate the accuracy of the recorded spot positions, the authors generated and executed a test treatment plan containing nine spot positions, to which the authors delivered ten MU each. The spot positions were measured with radiographic films and Matrixx 2D ion-chambers array placed at the isocenter plane and compared for displacements from the planned and recorded positions. Treatment logs for 14 patients were then used to determine the spot MU values and position accuracy of the scanning proton beam delivery system. Univariate analysis was used to detect any systematic error or large variation between patients, treatment dates, proton energies, gantry angles, and planned spot positions. The recorded patient spot positions and MU values were then used to replace the spot positions and MU values in the plan, and the treatment planning system was used to calculate the delivered doses to patients. The results were compared with the treatment plan. RESULTS: Within a treatment session, spot positions were reproducible within ±0.2 mm. The spot positions measured by film agreed with the planned positions within ±1 mm and with the recorded positions within ±0.5 mm. The maximum day-to-day variation for any given spot position was within ±1 mm. For all 14 patients, with ∼1 500 000 spots recorded, the total MU accuracy was within 0.1% of the planned MU values, the mean (x, y) spot displacement from the planned value was (-0.03 mm, -0.01 mm), the maximum (x, y) displacement was (1.68 mm, 2.27 mm), and the (x, y) standard deviation was (0.26 mm, 0.42 mm). The maximum dose difference between calculated dose to the patient based on the plan and recorded data was within 2%. CONCLUSIONS: The authors have shown that the treatment log file in a spot scanning proton beam delivery system is precise enough to serve as a quality assurance tool to monitor variation in spot position and MU value, as well as the delivered dose uncertainty from the treatment delivery system. The analysis tool developed here could be useful for assessing spot position uncertainty and thus dose uncertainty for any patient receiving spot scanning proton beam therapy.

A procedure to determine the planar integral spot dose values of proton pencil beam spots.

PURPOSE: Planar integral spot dose (PISD) of proton pencil beam spots (PPBSs) is a required input parameter for beam modeling in some treatment planning systems used in proton therapy clinics. The measurement of PISD by using commercially available large area ionization chambers, like the PTW Bragg peak chamber (BPC), can have large uncertainties due to the size limitation of these chambers. This paper reports the results of our study of a novel method to determine PISD values from the measured lateral dose profiles and peak dose of the PPBS. METHODS: The PISDs of 72.5, 89.6, 146.9, 181.1, and 221.8 MeV energy PPBSs were determined by area integration of their planar dose distributions at different depths in water. The lateral relative dose profiles of the PPBSs at selected depths were measured by using small volume ion chambers and were investigated for their angular anisotropies using Kodak XV films. The peak spot dose along the beam's central axis (D(0)) was determined by placing a small volume ion chamber at the center of a broad field created by the superposition of spots at different locations. This method allows eliminating positioning uncertainties and the detector size effect that could occur when measuring it in single PPBS. The PISD was then calculated by integrating the measured lateral relative dose profiles for two different upper limits of integration and then multiplying it with corresponding D(0). The first limit of integration was set to radius of the BPC, namely 4.08 cm, giving PISD(RBPC). The second limit was set to a value of the radial distance where the profile dose falls below 0.1% of the peak giving the PISD(full). The calculated values of PISD(RBPC) obtained from area integration method were compared with the BPC measured values. Long tail dose correction factors (LTDCFs) were determined from the ratio of PISD(full)∕PISD(RBPC) at different depths for PPBSs of different energies. RESULTS: The spot profiles were found to have angular anisotropy. This anisotropy in PPBS dose distribution could be accounted in a reasonable approximate manner by taking the average of PISD values obtained using the in-line and cross-line profiles. The PISD(RBPC) values fall within 3.5% of those measured by BPC. Due to inherent dosimetry challenges associated with PPBS dosimetry, which can lead to large experimental uncertainties, such an agreement is considered to be satisfactory for validation purposes. The PISD(full) values show differences ranging from 1 to 11% from BPC measured values, which are mainly due to the size limitation of the BPC to account for the dose in the long tail regions of the spots extending beyond its 4.08 cm radius. The dose in long tail regions occur both for high energy beams such as 221.8 MeV PPBS due to the contributions of nuclear interactions products in the medium, and for low energy PPBS because of their larger spot sizes. The calculated LTDCF values agree within 1% with those determined by the Monte Carlo (MC) simulations. CONCLUSIONS: The area integration method to compute the PISD from PPBS lateral dose profiles is found to be useful both to determine the correction factors for the values measured by the BPC and to validate the results from MC simulations.

Quantitative analysis of beam delivery parameters and treatment process time for proton beam therapy.

PURPOSE: To evaluate patient census, equipment clinical availability, maximum daily treatment capacity, use factor for major beam delivery parameters, and treatment process time for actual treatments delivered by proton therapy systems. METHODS: The authors have been recording all beam delivery parameters, including delivered dose, energy, range, spread-out Bragg peak widths, gantry angles, and couch angles for every treatment field in an electronic medical record system. We analyzed delivery system downtimes that had been recorded for every equipment failure and associated incidents. These data were used to evaluate the use factor of beam delivery parameters, the size of the patient census, and the equipment clinical availability of the facility. The duration of each treatment session from patient walk-in and to patient walk-out of the treatment room was measured for 82 patients with cancers at various sites. RESULTS: The yearly average equipment clinical availability in the last 3 yrs (June 2007-August 2010) was 97%, which exceeded the target of 95%. Approximately 2200 patients had been treated as of August 2010. The major disease sites were genitourinary (49%), thoracic (25%), central nervous system (22%), and gastrointestinal (2%). Beams have been delivered in approximately 8300 treatment fields. The use factor for six beam delivery parameters was also evaluated. Analysis of the treatment process times indicated that approximately 80% of this time was spent for patient and equipment setup. The other 20% was spent waiting for beam delivery and beam on. The total treatment process time can be expressed by a quadratic polynomial of the number of fields per session. The maximum daily treatment capacity of our facility using the current treatment processes was estimated to be 133 +/- 35 patients. CONCLUSIONS: This analysis shows that the facility has operated at a high performance level and has treated a large number of patients with a variety of diseases. The use factor of beam delivery parameters varies by disease site. Further improvements in efficiency may be realized in the equipment- and patient-related processes of treatment.

An MCNPX Monte Carlo model of a discrete spot scanning proton beam therapy nozzle.

PURPOSE: The purposes of this study were to validate a discrete spot scanning proton beam nozzle using the Monte Carlo (MC) code MCNPX and use the MC validated model to investigate the effects of a low-dose envelope, which surrounds the beam's central axis, on measurements of integral depth dose (IDD) profiles. METHODS: An accurate model of the discrete spot scanning beam nozzle from The University of Texas M. D. Anderson Cancer Center (Houston, Texas) was developed on the basis of blueprints provided by the manufacturer of the nozzle. The authors performed simulations of single proton pencil beams of various energies using the standard multiple Coulomb scattering (MCS) algorithm within the MCNPX source code and a new MCS algorithm, which was implemented in the MCNPX source code. The MC models were validated by comparing calculated in-air and in-water lateral profiles and percentage depth dose profiles for single pencil beams with their corresponding measured values. The models were then further tested by comparing the calculated and measured three-dimensional (3-D) dose distributions. Finally, an IDD profile was calculated with different scoring radii to determine the limitations on the use of commercially available plane-parallel ionization chambers to measure IDD. RESULTS: The distance to agreement, defined as the distance between the nearest positions of two equivalent distributions with the same value of dose, between measured and simulated ranges was within 0.13 cm for both MCS algorithms. For low and intermediate pencil beam energies, the MC simulations using the standard MCS algorithm were in better agreement with measurements. Conversely, the new MCS algorithm produced better results for high-energy single pencil beams. The IDD profile calculated with cylindrical tallies with an area equivalent to the area of the largest commercially available ionization chamber showed up to 7.8% underestimation of the integral dose in certain depths of the IDD profile. CONCLUSIONS: The authors conclude that a combination of MCS algorithms is required to accurately reproduce experimental data of single pencil beams and 3-D dose distributions for the scanning beam nozzle. In addition, the MC simulations showed that because of the low-dose envelope, ionization chambers with radii as large as 4.08 cm are insufficient to accurately measure IDD profiles for a 221.8 MeV pencil beam in the scanning beam nozzle.

Experimental characterization of the low-dose envelope of spot scanning proton beams.

In scanned proton beam radiotherapy, multiple pencil beams are used to deliver the total dose to the target volume. Because the number of such beams can be very large, an accurate dosimetric characterization of every single pencil beam is important to provide adequate input data for the configuration of the treatment planning system. In this work, we present a method to measure the low-dose envelope of single pencil beams, known to play a meaningful role in the dose computation for scanned proton beams. We measured the low-dose proton beam envelope, which extends several centimeters outwards from the center of each single pencil beam, by acquiring lateral dose profile data, down to relative dose levels that were a factor of 10(4) lower than the central axis dose. The overall effect of the low-dose envelope on the total dose delivered by multiple pencil beams was determined by measuring the dose output as a function of field size. We determined that the low-dose envelope can be influential even for fields as large as 20 cm x 20 cm.

Commissioning of the discrete spot scanning proton beam delivery system at the University of Texas M.D. Anderson Cancer Center, Proton Therapy Center, Houston.

PURPOSE: To describe a summary of the clinical commissioning of the discrete spot scanning proton beam at the Proton Therapy Center, Houston (PTC-H). METHODS: Discrete spot scanning system is composed of a delivery system (Hitachi ProBeat), an electronic medical record (Mosaiq V 1.5), and a treatment planning system (TPS) (Eclipse V 8.1). Discrete proton pencil beams (spots) are used to deposit dose spot by spot and layer by layer for the proton distal ranges spanning from 4.0 to 30.6 g/cm2 and over a maximum scan area at the isocenter of 30 x 30 cm2. An arbitrarily chosen reference calibration condition has been selected to define the monitor units (MUs). Using radiochromic film and ion chambers, the authors have measured spot positions, the spot sizes in air, depth dose curves, and profiles for proton beams with various energies in water, and studied the linearity of the dose monitors. In addition to dosimetric measurements and TPS modeling, significant efforts were spent in testing information flow and recovery of the delivery system from treatment interruptions. RESULTS: The main dose monitors have been adjusted such that a specific amount of charge is collected in the monitor chamber corresponding to a single MU, following the IAEA TRS 398 protocol under a specific reference condition. The dose monitor calibration method is based on the absolute dose per MU, which is equivalent to the absolute dose per particle, the approach used by other scanning beam institutions. The full width at half maximum for the spot size in air varies from approximately 1.2 cm for 221.8 MeV to 3.4 cm for 72.5 MeV. The measured versus requested 90% depth dose in water agrees to within 1 mm over ranges of 4.0-30.6 cm. The beam delivery interlocks perform as expected, guarantying the safe and accurate delivery of the planned dose. CONCLUSIONS: The dosimetric parameters of the discrete spot scanning proton beam have been measured as part of the clinical commissioning program, and the machine is found to function in a safe manner, making it suitable for patient treatment.

The M. D. Anderson proton therapy system.

PURPOSE: The purpose of this study is to describe the University of Texas M. D. Anderson proton therapy system (PTC-H) including the accelerator, beam transport, and treatment delivery systems, the functionality and clinical parameters for passive scattering and pencil beam scanning treatment modes, and the results of acceptance tests. METHODS: The PTC-H has a synchrotron (70-250 MeV) and four treatment rooms. An overall control system manages the treatment, physics, and service modes of operation. An independent safety system ensures the safety of patients, staff, and equipment. Three treatment rooms have isocentric gantries and one room has two fixed horizontal beamlines, which include a large-field treatment nozzle, used primarily for prostate treatments, and a small-field treatment nozzle for ocular treatments. Two gantry treatment rooms and the fixed-beam treatment room have passive scattering nozzles. The third gantry has a pencil beam scanning nozzle for the delivery of intensity modulated proton treatments (IMPT) and single field uniform dose (SFUD) treatments. The PTC-H also has an experimental room with a fixed horizontal beamline and a passive scattering nozzle. The equipment described above was provided by Hitachi, Ltd. Treatment planning is performed using the Eclipse system from Varian Medical Systems and data management is handled by the MOSAIQ system from IMPAC Medical Systems, Inc. The large-field passive scattering nozzles use double scattering systems in which the first scatterers are physically integrated with the range modulation wheels. The proton beam is gated on the rotating range modulation wheels at gating angles designed to produce spread-out-Bragg peaks ranging in size from 2 to 16 g/cm2. Field sizes of up to 25 x 25 cm2 can be achieved with the double scattering system. The IMPT delivery technique is discrete spot scanning, which has a maximum field size of 30 x 30 cm2. Depth scanning is achieved by changing the energy extracted from the synchrotron (energy can be changed pulse to pulse). The PTC-H is fully integrated with DICOM-RT ION interfaces for imaging, treatment planning, data management, and treatment control functions. RESULTS: The proton therapy system passed all acceptance tests for both passive scattering and pencil beam scanning. Treatments with passive scattering began in May 2006 and treatments with the scanning system began in May 2008. The PTC-H was the first commercial system to demonstrate capabilities for IMPT treatments and the first in the United States to treat using SFUD techniques. The facility has been in clinical operation since May 2006 with up-time of approximately 98%. CONCLUSIONS: As with most projects for which a considerable amount of new technology is developed and which have duration spanning several years, at project completion it was determined that several upgrades would improve the overall system performance. Some possible upgrades are discussed. Overall, the system has been very robust, accurate, reproducible, and reliable. The authors found the pencil beam scanning system to be particularly satisfactory; prostate treatments can be delivered on the scanning nozzle in less time than is required on the passive scattering nozzle.

Effects of long-term administration of royal jelly on pituitary weight and gene expression in middle-aged female rats.

To determine the effects of ingested royal jelly (RJ) on the pituitary in middle-aged female rats, we performed a long-term RJ administration test. Several animals showed age-related increases in pituitary weight, and RJ administration compensated for the increase. RJ tended to down-regulate prolactin mRNA and up-regulated thyroid-stimulating hormone beta mRNA in the pituitary. This suggests that RJ compensates for age-associated decline in pituitary functions.

Estrogenic activities of Fatty acids and a sterol isolated from royal jelly.

We have previously reported that royal jelly (RJ) from honeybees (Apis mellifera) has weak estrogenic activity mediated by interaction with estrogen receptors that leads to changes in gene expression and cell proliferation. In this study, we isolated four compounds from RJ that exhibit estrogenic activity as evaluated by a ligand-binding assay for the estrogen receptor (ER) beta. These compounds were identified as 10-hydroxy-trans-2-decenoic acid, 10-hydroxydecanoic acid, trans-2-decenoic acid and 24-methylenecholesterol. All these compounds inhibited binding of 17beta-estradiol to ERbeta, although more weakly than diethylstilbestrol or phytoestrogens. However, these compounds had little or no effect on the binding of 17beta-estradiol to ERalpha. Expression assays suggested that these compounds activated ER, as evidenced by enhanced transcription of a reporter gene containing an estrogen-responsive element. Treatment of MCF-7 cells with these compounds enhanced their proliferation, but concomitant treatment with tamoxifen blocked this effect. Exposure of immature rats to these compounds by subcutaneous injection induced mild hypertrophy of the luminal epithelium of the uterus, but was not associated with an increase in uterine weight. These findings provide evidence that these compounds contribute to the estrogenic effect of RJ.

A dipeptide YY derived from royal jelly proteins inhibits renin activity.

Renin is the rate limiting enzyme in the renin-angiotensin (RA) system that regulates blood pressure and electrolyte balance. In this study, we investigated the renin inhibitory effect of a royal jelly (RJ)-derived peptide. A dipeptide YY was isolated from the digested fraction of RJ proteins by proteases and was found to inhibit human renin activity. The inhibition constant (Ki) of YY was estimated to be 10 microM when the Km was 0.16 microM using sheep angiotensinogen as the substrate. The peptide was observed to lower blood pressure in spontaneously hypertensive rats.

Royal jelly stimulates bone formation: physiologic and nutrigenomic studies with mice and cell lines.

Royal jelly (RJ) has diverse physiological and pharmacological functions. We observed its weak estrogenic activity in the previous study. RJ stimulated the proliferation of mouse osteoblast-like MC3T3-E1 cells at 0.1 mg/ml, and the effect was blocked by the specific estrogen receptor antagonist ICI 182,780. The addition of 0.1-1.0 mg/ml RJ enhanced collagen production in culture medium. Oral administration of RJ to normal female mice for 9 weeks increased the ash content of their tibiae. DNA microarray analysis revealed significant changes in gene expression related to extracellular matrix formation when the femurs of mice fed RJ were analyzed. Quantitative reverse transcriptase-PCR (RT-PCR) confirmed up-regulation of procollagen I alpha1 gene expression. These data suggest that RJ as a whole or some of its individual components stimulates production of type I collagen and other activities for bone formation through action on osteoblasts.

Identification of caffeoylquinic acid derivatives from Brazilian propolis as constituents involved in induction of granulocytic differentiation of HL-60 cells.

We have previously reported that Brazilian propolis extracts inhibited growth of HL-60 human myeloid leukemia cells, which is partly attributed to the induction of apoptosis associated with granulocytic differentiation. In this study, we isolated three compounds which induce granulocytic differentiation evaluated by nitroblue tetrazolium (NBT)-reducing assays from the water extract of propolis and identified as 4,5-di-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, and 3,4-di-O-caffeoylquinic acids by NMR analysis. Cell growth inhibitory activity of these caffeoylquinic acids was found in HL-60 cell, which was mainly attributed to the induction of apoptosis. Furthermore, the potency of caffeoylquinic acid derivatives to induce granulocytic differentiation was examined in HL-60 cells. Caffeic, quinic, and chlorogenic acids had no effects on the NBT-reducing activity, while 3,4,5-tri-O-caffeoylquinic acid induced more than 30% of NBT-positive cells. These results suggest that the number of the caffeoyl groups bound to quinic acid plays an important role in the potency of the caffeoylquinic acid derivatives to induce granulocytic differentiation. This is the first report demonstrating that the caffeoylquinic acid derivatives induce granulocytic differentiation of HL-60 cells.

Royal jelly has estrogenic effects in vitro and in vivo.

Royal jelly (RJ) from honeybees (Apis mellifera) is traditionally thought to improve menopausal symptoms. The potential estrogenic activities of RJ were investigated using various approaches. RJ competed for binding of 17beta-estradiol to the human estrogen receptor alpha and beta but its affinities were weak compared with diethylstilbestrol and phytoestrogens. The reporter gene expression assays suggested that 0.1-1 mg/ml RJ activated estrogen receptors, leading to enhanced transcription of a reporter gene through an estrogen-responsive element. 1 mg/ml RJ stimulated the mRNA expression of estrogen-responsive pS2 and vascular endothelial growth factor (VEGF) by increasing gene transcription in MCF-7 cells. Treatment with RJ at concentrations ranging from 0.5 to 1 mg/ml enhanced MCF-7 cell proliferation, but concomitant treatment with 1 microM tamoxifen blocked this effect. In vivo studies using ovariectomized rats showed that 17beta-estradiol (20 mg/kg, s.c.) treatment restored VEGF expression in both uterus and brain, whereas RJ (1 g/kg, s.c.) restored it in uterus but not in brain. These findings provide evidence that RJ has estrogenic activities through interaction with estrogen receptors followed by endogenous gene expressions.

Effects of propolis on cell growth and gene expression in HL-60 cells.

Brazilian propolis obtained from honeybee hives was extracted with water or ethanol. Cell growth-inhibitory activities of these propolis extracts were found in HL-60 human myeloid leukemia cells. The extracts-induced apoptosis in the cells, which was characterized by morphological and nucleosomal DNA fragmentation analysis. The apoptosis was mainly attributed to the induction of granulocytic differentiation, which was evaluated by nitro blue tetrazolium (NBT) reducing assays and cytofluorometric analysis for the expression of cell surface marker CD11b. DNA microarray analysis was performed to examine the gene expression profiles in the propolis-treated HL-60 cells accompanied with granulocytic differentiation, which were compared with those in all-trans retinoic acid-treated cells. Several genes were up- or down-regulated. Two genes encoding S100 calcium binding protein A9 and ferritin, heavy polypeptide 1 were up-regulated, which were also confirmed by semi-quantitative reverse transcriptase-PCR (RT-PCR). Propolis-induced growth inhibition in HL-60 cells was, at least in part, due to differentiation with gene expression profiles, which are similar to those induced by all-trans retinoic acid.

Antihypertensive effect of peptides from royal jelly in spontaneously hypertensive rats.

We have shown that Protease N treated Royal Jelly (ProRJ) and peptides from ProRJ (Ile-Tyr (IY), Val-Tyr (VY), Ile-Val-Tyr (IVY)) inhibited angiotensin I-converting enzyme (ACE) activity and they have an antihypertensive effect in repeated oral administration for 28 d on spontaneously hypertensive rats (SHR). We investigated the contributive ratio of these peptides in ProRJ for antihypertensive effect in single oral administration on SHR. In single oral administration of each peptide and peptides mixture (MIX; IY, VY and IVY) at doses of 0.5, 1 and 10 mg/kg, systolic blood pressure (SBP) of SHR was reduced dose-dependently. This antihypertensive effect was held for 8 h. These results suggest that peptides contributed to the antihypertensive effect of ProRJ. And the contributive ratio of MIX in ProRJ for antihypertensive effect was computed to be about 38%. Therefore it is considered that intake of peptides, as a functional food would be beneficial for improving blood pressure in people with hypertension.