Data pertaining to aberrant intracellular calcium handling during androgen deprivation therapy in prostate cancer
The data generated here in relates to the research article “CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer”. Our hypothesis was that CaV1.3 contributed to altered calcium during androgen deprivation therapy (ADT) in prostate cancer, leading to castrate resistance disease. Here a model of prostate cancer (PCa) progression to castration resistance was employed, with untreated androgen sensitive LNCaP cell line alongside two androgens deprived (bicalutamide) sublines, either 10 days (LNCaP-ADT) or 2 years (LNCaP-ABL) treatment. Within which using qPCR we examined fold change gene expression of markers linked to androgen resistance, androgen receptor (AR) and neuron specific enolase (NSE), observing an increase under ADT. In addition, the gene expression of a range of calcium channels was measured, with the L-type Voltage gated calcium channel, CaV1.3, demonstrating the largest increase during ADT compared to DMSO control. The effect of CaV1.3 on the gene expression of calcium channels Orai and STIM1 was tested using CaV1.3 siRNA knockdown, with no effect observed. Protein fractionation and subsequent CaV1.3 western blot noted the presence of CaV1.3 c-terminus in the nucleus which was lost in LNCaP-ABL. The calcium channel blocker (CCB), nifedipine, was employed to determine the impact of CaV1.3 on store release and calcium entry measured with Fura-2AM ratiometric dye. In both the presence and absence ADT, nifedipine was found to have no impact on store release induced by thapsigargin (Tg) in 0mM Ca2+ nor calcium entry following the addition of 2mM Ca2. The effect of nifedipine on CaV1.3 in PCa biology was measured through cell survival colony formation assay, with no observed change in the presence of CCB. This data can be reused to inform new studies investigating altered calcium handling in androgen resistant prostate cancer. It provides insight into the mechanism of CaV1.3 and its functional properties in altered calcium in cancer, which can be of use to researchers investigating this channel in disease. Furthermore, it could be helpful in interpretting studies investigating CCB’s as a therapeutic and also in the development of future drugs targeting CaV1.3.
Steps to reproduce
Tissue Culture - LNCaP (ATCC # CRL-1740). 10µM bicalutamide (Sigma) LNCaP-ADT 10day treated. 2year ADT insensitive CRPC, LNCaP-ABL. Maintained in RPMI (Gibco) with 10% fetal bovine serum (Gibco) at 37oC and 5% CO2. Nifedipine final concentration10µM. siRNA transfection - Dharmacon transfection reagent along with Dharmacon ON-TARGETplus siRNA Non-targeting and CACNA1D siRNA. Cells were incubated at 37oC with 5% CO2 for 48 hours prior to RNA extraction. RNA extraction and cDNA synthesis- Total ribonucleic acid (RNA) extracted with High Pure RNA Isolation Kit (Roche). Quantified using NanoDrop 1000™ (Thermo Scientific). Complimentary deoxyribonucleic acid (cDNA) using Transcriptor first strand cDNA synthesis (Roche). Real Time Polymerase Chain Reaction (qPCR) - LightCycler Nano™ (Roche) was programmed to run as outlined in the FastStart Essential DNA Green Master protocol, annealing temperature as per primer melting temperature. Housekeeping gene HPRT1 was used. Cellular fractionation was done using a kit (Abcam) with approximately 6*106 cells per sample. Western Blot – Protein lysates were separated on a Bolt Bis-Tris Plus pre-cast Gels (4-12%, Invitrogen) using the Mini Gel Tank (Invitrogen). 50µg total protein, denatured at 70oC for 10mins. Samples were loaded into the tank filled with x1 Bolt MOPS SDS running buffer (Invitrogen), run at 200V for 30mins. Transferred to a PVDF membrane 0.4µm in Transfer buffer (Bolt Transfer buffer (Invitrogen) with Bolt Antioxidant (Invitrogen) at 20V for 60mins. Incubated in blocking buffer (Tris buffered saline, 0.1% tween 20 5% milk) 1h at RTP, three 5 min washes in TBST. Incubated with CaV1.3 C-terminus antibody (Abcam, 1:500 dilution, AB-84811) TBST 5% milk over night at 4oC. TBST wash before 1hr incubation HRP-Anti Mouse (HRP-Anti-Mouse, BD Pharmigen,1:1000). Developed with 1:4 Supersignal West Dura Chemiluminescent Substrate (Thermo Scientific) for 1min at RTP. Exposed to a piece of X-ray film in cassette and placed in developer solution. Calcium Measurements - Cells were resuspended in OptiMEM™ low serum media at a concentration of 5*105 /ml. Fura 2-AM was added to the tube to a final concentration of 2uM and incubated in dark RT 45mins. The pellet was resuspended in 500µl physiological saline solution PSS 0% Ca2+ (NaCl 142mM, MgCl2 1mM, KCl 4mM, D-glucose 11.1mM,1 mM EGTA and HEPES 10mM pH balanced to 7.4 with NaOH). 100µl was added to each well of black walled 96 well plate to give 1*105 cells/well. Measurements made using VICTOR multilabel plate reader, excitation wavelengths 340 and 380nm and emission wavelength 510nm recorded every 5s. Baseline Ca2+ levels were determined for 100s then Tg was injected at final concentration of 4µM and recorded for 400s after which CaCl2 was injected at a final concentration of 2mM with measurements recorded for a further 200s. Cell Proliferation was assayed using a Wst-1 assay kit (Roche) in a VICTOR multilabel plate reader, absorbance recorded at 440nm.