Effects Of Androgen On Intracellular Free Calcium Concentration Of Prostate Cancer Cells And Its Underlying Mechanism

Background The appropriate regulation of androgen activity is necessary fora range of developmental and physiological processes, particularly male sexualdevelopment and maturation, as well as the maintenance of male reproductive organsand of spermatogenesis . The principle steroidal androgens, testosterone (T) and itsmetabolite 5-α dihydrotestosterone (DHT), are thought to predominantly mediate theirbiological effects through binding to the androgen receptor (AR). AR, in commonwith other members of the nuclear receptor superfamily, functions as aligand-inducible transcription factor. The binding of T or DHT to AR inducesreceptor dimerization, facilitating the ability of AR to bind to its cognate responseelement and recruit coregulators to promote the expression of target genes .This is thebasis of prostate cancer's hormonal therapy. In addition to this transcriptional orgenomic mode of action by steroids, an increasing body of evidence suggests thatandrogens, like progesterone and estrogen, can exert rapid, nongenomic effects.Nongenomic steroid activity typically involves the rapid induction of conventionalsecond messenger signal transduction cascades, including increases in freeintracellular calcium. Ca2+ functions as an ubiquitous second messenger andmodulation of intracellular Ca2+ levels regulates a wide range of cellular processes,including proliferation, apoptosis, motility, and gene expression. The elevation ofintracellular Ca2+ is detected by specific Ca 2+sensor molecules, including PKC andcalmodulin (CaM), to induce signal transduction cascades and modulation oftranscription factor activity. The specific cellular response to elevation of Ca2+ levelsis highly dependent of the strength and duration of the Ca2+.Thus, it is verysignificant for the therapy of prostate cancer to evaluate the effects of androgen onintracellular calcium of prostate cancer cells. Objective The effect of androgen (5α-dihydrotestosterone, DHT) onintracellular Ca2+ concentrations ([Ca2+]i) in hormone-sensitive LNCaP humanprostate cancer cells was examined, and investigated the mechanism. Methods LNCaP cells were grown in normal RPMI 1640 medium - 4 -硕士毕业论文 英文摘要supplemented with10% FBS. cells were incubated for 2–3 days at 37oC in ahumidified humidified atmosphere of 5% CO2 before they were used formeasurements. Cultures were washed twice with Krebs-HEPEs. Cells were loadedwith the calcium-sensitive dyes by incubation with 3μM Fluo-2/AM and 0.04%pluronic F-127 for 30 minutes in the Krebs-HEPEs at 37oC. Subsequently, the cultureswere washed three times with Krebs-HEPEs and the carrying cells was mounted in afluorescence microscope. After each experiment, the maximum fluorescence waschecked with the introduction of 10-5M ionomycin. Any fura-2 leakage, the maximumand the minimum fluorescence (Fmax and Fmin) were determined by the addition of10-5M ionomycin ,10-2M CaCl2,and 10-2M EGTA to cuvettes. The fluorescenceemission ratios at 510 nm were determined following alternating excitation at 340 and380nm. Calcium concentrations were calculated based on calibration curves that wereconstucted from the fluorescence ratios (340nm/380nm) of solutions whichcontained Krebs-HEPES or calcium-free Krebs-HEPES. Results Rapid increase in calcium levels were found following addition ofDHT.the latency of response is only in seconds. DHT at the concentration of 10-9M,10-8M,10-7M , 10-6M respectively elevated [Ca2+]i from 28士5nM,29土5nM,28士4nM and 28士9 nM to 31土3 nM (P>0.05) , 95士135nM ( P <0. 01),193士335nM(P<0. 001) and 208 士 425nM (P<0.001)respectively. when extracelluar fluidcontained zero calcium, or LNCaP cells was treated with several blocks of L-typevoltage-gated calcium channels including verapamil, diltiazem , and nifedipine at 37oCfor 5 min prior to stimulation with DHT at...