Vitamin D Favorably Alters the Cancer Promoting Prostaglandin Cascade

Patients and Methods

Participants. Candidates were enrolled in an institutional review board-approved protocol (ClinicalTrials.gov Identifier NCT01769625). Prior to enrollment and at the end of the study, participants had serum chemistries and complete blood count drawn to insure normalcy and no change related to treatment. Participants had to lack a history which would make them at high risk (≥1.67% at 5 years) for breast cancer, had to not have taken nonsteroidal anti-inflammatory medications within two weeks of enrollment, nor while on study (except celecoxib if randomized to receive this). They could not be pregnant. A urine pregnancy test was to be obtained within 48 h of enrollment unless the candidate had not had a menstrual cycle in the last 12 months, she was >55 years old, or had undergone hysterectomy. Of 36 individuals who enrolled, eight withdrew before completion: five elected not to continue, two developed cold-like symptoms, one a rash. Participants were randomized in double blind fashion to one of four treatment groups for one month: placebo, 400 IU or 2,000 IU vitamin D-3 daily, or 2,000 IU vitamin D-3 plus 400 mg celecoxib daily. Vitamin D-3 doses were obtained from Tishcon (Westbury, NY, USA); celecoxib was obtained from Pfizer (New York, NY, USA). Due to low enrollment, with potential participants indicating they did not want to enroll if they might receive placebo, the protocol was revised such that additional recruits were randomized to one of three groups which included vitamin D-3.

Sample collection. We asked permission to collect three sample types from each participant: blood, breast nipple aspirate fluid (NAF), and mammary ductoscopy (MD) samples. NAF collection was attempted from at least one breast, and if agreed, MD samples were collected from the same breast both before and after treatment. Scientists performing the biomarker analyses were blinded as to the participant's treatment assignment. Participants were asked to take their last dose of medication the morning of collection. All samples were collected within six hours of their last medication dose.

Blood was collected and serum or plasma separated using standard clinical laboratory techniques. NAF was aspirated by a trained physician or nurse clinician using a modified breast pump (7). After nipple aspiration was completed and if the participant agreed, a topical anesthetic was applied to the nipple. This was followed by a nipple block with injected lidocaine. Whenever possible, the same duct from which NAF was collected was entered with a mammary ductoscope (Solos Endoscopy, Boston, MA, USA).

Sample analysis. PGE₂: Each NAF and serum specimen and standard was be analyzed in duplicate. Because of variability in the concentration of biomolecules, NAF PGE₂ biomarker levels were calculated per mg of total protein. Total protein in NAF was analyzed using a Pierce BCA Kit (Rockford, IL, USA). PGE₂ was measured using the high sensitivity PGE₂ immunoassay kit from Oxford Biomedical Research (Oxford, MI, USA) per manufacturer's instructions.

25(OH)D: Serum levels of 25(OH)D-2, 25(OH)D-3, and total 25(OH)D were determined as previously described (8). Briefly, liquid chromatography-mass spectrometry (LC-MS/MS) was performed to determine the contribution of 25(OH)D-2 and 25(OH)D-3 to serum 25(OH)D using a TSQ Quantum Ultra triple mass spectrometer (Thermo Finnigan Corp., San Jose, CA, USA).

Celecoxib: High performance liquid chromatography (HPLC) MS analysis was performed using an Agilent 1100 Series with an G1969 high resolution time of flight (TOF) MS system with an electropray (ESI) source (Agilent, Santa Clara, CA, USA). The chromatographic and mass spectral data were acquired using MassHunter software (Agilent). Samples were purified using solid-phase extraction (SPE) with Bond Elute C₁₈ cartridges.

TGFβ1 and -2: The concentrations of TGFβ1 and β2 in NAF and serum samples were measured by an immunoassay kit from R&D Systems (Minneapolis, MN, USA), following the manufacturer's instructions. The samples were analyzed in duplicate.

COX2: Total RNA was extracted from the duct samples with a kit from Qiagen (Valencia, CA, USA) following the instructions of the manufacturer. Twelve microliters of eluted RNA were reverse-transcribed with the Reverse Transcription (RT) Kit from Qiagen (Cat# 205311) according to the manufacturer instructions. For real-time PCR, 5 μl diluted RT products were included in 20 μl reactions. The primers, probes, and TaqMan® Gene Expression Master Mix kit (Cat# 4369016) were purchased from Life Technologies (Carlsbad, CA, USA) to analyze of expressions of COX2 and GAPDH in the duct samples. All reactions were run on a MyiQ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 45 s. GAPDH served as an internal control for normalization of the data.

Statistical analysis. The change in biomarkers after treatment was calculated for each variable by subtracting the initial values from the final. Extreme values, including all values greater or less than two standard deviations from the mean were considered outliers and removed. All biomarker values were natural log-transformed prior to analysis. Comparisons of changes with treatment for each biomarker were conducted using one-way analysis of variance (ANOVA). A post-hoc comparison was performed applying Bonferroni's correction to evaluate the interactions between the four intervention groups and to identify the groups with significant differences. For all biomarkers, univariate one-way analysis of covariance (ANCOVA) was used to determine whether any of the measured demographic variables influenced the results. All determinations include the assessment of confounding by demographic variables, with an acceptable level of significance set at p<0.05. Descriptive statistics were also determined for each of the variables.