Mipsagargin, a novel thapsigargin-based PSMA-activated prodrug: results of a first-in-man phase I clinical trial in patients with refractory, advanced or metastatic solid tumours.

BACKGROUND: Mipsagargin (G-202; (8-O-(12-aminododecanoyl)-8-O-debutanoyl thapsigargin)-Asp-γ-Glu-γ-Glu-γ-GluGluOH)) is a novel thapsigargin-based targeted prodrug that is activated by PSMA-mediated cleavage of an inert masking peptide. The active moiety is an inhibitor of the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump protein that is necessary for cellular viability. We evaluated the safety of mipsagargin in patients with advanced solid tumours and established a recommended phase II dosing (RP2D) regimen. METHODS: Patients with advanced solid tumours received mipsagargin by intravenous infusion on days 1, 2 and 3 of 28-day cycles and were allowed to continue participation in the absence of disease progression or unacceptable toxicity. The dosing began at 1.2 mg m(-2) and was escalated using a modified Fibonacci schema to determine maximally tolerated dose (MTD) with an expansion cohort at the RP2D. Plasma was analysed for mipsagargin pharmacokinetics and response was assessed using RECIST criteria. RESULTS: A total of 44 patients were treated at doses ranging from 1.2 to 88 mg m(-2), including 28 patients in the dose escalation phase and 16 patients in an expansion cohort. One dose-limiting toxicity (DLT; Grade 3 rash) was observed in the dose escalation portion of the study. At 88 mg m(-2), observations of Grade 2 infusion-related reaction (IRR, 2 patients) and Grade 2 creatinine elevation (1 patient) led to declaration of 66.8 mg m(-2) as the recommended phase II dose (RP2D). Across the study, the most common treatment-related adverse events (AEs) were fatigue, rash, nausea, pyrexia and IRR. Two patients developed treatment-related Grade 3 acute renal failure that was reversible during the treatment-free portion of the cycle. To help ameliorate the IRR and creatinine elevations, a RP2D of 40 mg m(-2) on day 1 and 66.8 mg m(-2) on days 2 and 3 with prophylactic premedications and hydration on each day of infusion was established. Clinical response was not observed, but prolonged disease stabilisation was observed in a subset of patients. CONCLUSIONS: Mipsagargin demonstrated an acceptable tolerability and favourable pharmacokinetic profile in patients with solid tumours.

LSD1 inhibition suppresses ASCL1 and de-represses YAP1 to drive potent activity against neuroendocrine prostate cancer.

Lysine-specific demethylase 1 (LSD1 or KDM1A ) has emerged as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Among mCRPC subtypes, neuroendocrine prostate cancer (NEPC) is an exceptionally aggressive variant driven by lineage plasticity, an adaptive resistance mechanism to androgen receptor axis-targeted therapies. Our study shows that LSD1 expression is elevated in NEPC and associated with unfavorable clinical outcomes. Using genetic approaches, we validated the on-target effects of LSD1 inhibition across various models. We investigated the therapeutic potential of bomedemstat, an orally bioavailable, irreversible LSD1 inhibitor with low nanomolar potency. Our findings demonstrate potent antitumor activity against CRPC models, including tumor regressions in NEPC patient-derived xenografts. Mechanistically, our study uncovers that LSD1 inhibition suppresses the neuronal transcriptional program by downregulating ASCL1 through disrupting LSD1:INSM1 interactions and de-repressing YAP1 silencing. Our data support the clinical development of LSD1 inhibitors for treating CRPC - especially the aggressive NE phenotype. Statement of Significance: Neuroendocrine prostate cancer presents a clinical challenge due to the lack of effective treatments. Our research demonstrates that bomedemstat, a potent and selective LSD1 inhibitor, effectively combats neuroendocrine prostate cancer by downregulating the ASCL1- dependent NE transcriptional program and re-expressing YAP1.

A phase I/IIA study of AGS-PSCA for castration-resistant prostate cancer.

BACKGROUND: This first-in-human phase I/IIA study was designed to evaluate the safety and pharmacokinetics (PKs) of AGS-PSCA a fully human monoclonal antibody directed to prostate stem cell antigen (PSCA) in progressive castration-resistant prostate cancer. PATIENTS AND METHODS: Twenty-nine patients were administered infusions of AGS-PSCA (1-40 mg/kg) every 3 weeks for 12 weeks; 18 final patients received a 40-mg/kg loading dose followed by 20-mg/kg repeat doses. Primary end points were safety and PK. Immunogenicity, antitumor activity and circulating tumor cells were also evaluated. RESULTS: No drug-related serious adverse events were noted. Dose escalation stopped before reaching the maximum tolerated dose as target concentrations were achieved. Drug levels accumulated linearly with dose and the mean terminal half-life was 2-3 weeks across dose levels. The 40-mg/kg loading dose followed by repeated 20-mg/kg doses yielded serum drug concentrations above the projected minimum therapeutic threshold after two to three doses without excessive drug accumulation or toxicity. Significant antitumor effects were not seen. CONCLUSIONS: A 40-mg/kg loading dose followed by 20-mg/kg infusions every 3 weeks is the recommended phase II dose of AGS-PSCA. PSCA is a promising drug target and studies in prostate and other relevant solid tumors are planned.

Nutritional status of long-term patients in the acute care setting.

The nutritional status of 926 patients (51.4% female) at an acute tertiary private hospital with a length of stay ≥14 days was assessed using Subjective Global Assessment. The prevalence of malnutrition was 42.5% (37.2% length of stay of 14-27 days, 51.6% ≥28 days). From logistic regression analysis, length of stay and age were independent predictors of malnutrition. It is important that the nutritional status of longer stay patients is monitored and appropriate nutrition support is commenced.

Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors.

Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.

Combinatorial androgen receptor targeted therapy for prostate cancer.

Prostatic carcinogenesis is associated with changes in the androgen receptor (AR) axis converting it from a paracrine dependence upon stromal signaling to an autocrine-initiated signaling for proliferation and survival of prostatic cancer cells. This malignant conversion is due to gain of function changes in which the AR activates novel genomic (i.e. transcriptional) and non-genomic signaling pathways, which are not present in normal prostate epithelial cells. During further progression, additional molecular changes occur which allow these unique malignancy-dependent AR signaling pathways to be activated even in the low androgen ligand environment present following androgen ablation therapy. These signaling pathways are the result of partnering the AR with a series of other genomic (e.g. transcriptional co-activators) or non-genomic (e.g. steroid receptor co-activator (Src) kinase) signaling molecules. Thus, a combinatorial androgen receptor targeted therapy (termed CART therapy) inhibiting several points in the AR signaling cascade is needed to prevent the approximately 30,000 US males per year dying subsequent to failure of standard androgen ablation therapy. To develop such CART therapy, a series of agents targeted at specific points in the AR cascade should be used in combination with standard androgen ablative therapy to define the fewest number of agents needed to produce the maximal therapeutic anti-prostate cancer effect. As an initial approach for developing such CART therapy, a variety of new agents could be combined with luteinizing hormone-releasing hormone analogs. These include: (1) 5alpha-reductase inhibitors to inhibit the conversion of testosterone to the more potent androgen, dihydrotestosterone; (2) geldanamycin analogs to downregulate AR protein in prostate cancer cells, (3) 'bulky' steroid analogs, which can bind to AR and prevent its partnering with other co-activators/signaling molecules, and (4) small molecule kinase inhibitors to inhibit MEK, which is activated as part of the malignant AR signaling cascade.

Enzymatic activation of a doxorubicin-peptide prodrug by prostate-specific antigen.

New approaches to target cytotoxic therapy specifically to metastatic prostate cancer sites are urgently needed. As such an approach, an inactive prodrug was synthesized by coupling the primary amine of doxorubicin to the COOH-terminal carboxyl of a seven-amino acid peptide carrier (i.e., Mu-His-Ser-Ser-Lys-Leu-Gln-Leu). The seven-amino acid peptide was documented to be hydrolyzable specifically by the serine protease prostate-specific antigen (PSA) to liberate the active cytotoxin L-leucyl-doxorubicin. Primary cultures of PC-82 human prostate cancer cells secreted high levels of enzymatically active PSA (i.e., 70 +/- 5 ng of enzymatically active PSA/10(6) cells/24 h), whereas LNCaP human prostate cancer cells produced lower levels of enzymatically active PSA (i.e., 2.3 +/- 1 ng/10(6) cells/24 h). LNCaP cells, however, secreted sufficient amounts of enzymatically active PSA to activate the doxorubicin prodrug to a cytotoxic form in vitro. The specificity of the cytotoxic response to the prodrug was demonstrated by the fact that 70 nM of the prodrug killed 50% of the PSA-producing LNCaP cells, whereas doses as high as 1 microM had no cytotoxic effect on PSA-nonproducing TSU human prostate cancer cells in vitro.

Specific and efficient peptide substrates for assaying the proteolytic activity of prostate-specific antigen.

Prostate-specific antigen (PSA) is a serine protease secreted by both normal prostate glandular cells and prostate cancer cells. The major proteolytic substrates for PSA are the gel-forming proteins in semen, semenogelin (Sg) I and II. On the basis of the PSA cleavage map for Sg I and II, a series of small peptides (i.e., < or = 7 amino acids) was synthesized and coupled at the COOH terminus to 7-amino-4-methyl coumarin. Using these fluorescently tagged substrates, K(m)s and k(cat)s were determined for PSA hydrolysis, and the substrates were also tested for activity against a panel of purified proteases. Previously, a variety of chymotrypsin substrates have been used to assay the enzymatic activity of PSA. The present studies have identified a peptide sequence with a high degree of specificity for PSA (ie., no detectable hydrolysis by chymotrypsin) and improved K(m)s and k(cat)s over previously used substrates. On the basis of these parameters, the best peptide substrate for PSA has the amino acid sequence HSSKLQ. Using PC-82 human prostate cancer xenografts and human prostate tissues, this PSA substrate was used to document that prostate cancer cells secrete enzymatically active PSA into the extracellular fluid but that once in the blood, PSA is not enzymatically active. On the basis of this information, it should be possible to use the HSSKLQ peptide as a carrier to target peptide-coupled prodrugs for selective activation within sites of PSA-secreting, metastatic prostate cancer cells and not within the blood or other nonprostatic normal tissues.

The influence of prednisone on the efficacy of docetaxel in men with metastatic castration-resistant prostate cancer.

BACKGROUND: Prednisone and other corticosteroids can provide palliation and tumor responses in patients with prostate cancer. The combination of docetaxel and prednisone was the first treatment shown to prolong survival in men with metastatic castration-resistant prostate cancer (mCRPC). Since the approval of docetaxel in 2004, additional treatments are available, including abiraterone, which is also administered with prednisone. Therefore, patients are increasingly likely to have prednisone therapy several times throughout their disease course, and the contribution of prednisone to the efficacy of docetaxel is unknown. METHODS: We conducted a retrospective study of patients with mCPRC treated with docetaxel at our institution between 2004 and 2014. Patients were divided into two cohorts based upon whether prednisone was co-administered with docetaxel. Cohorts were further stratified based upon prior prednisone (with abiraterone) or hydrocortisone (with ketoconazole) use. The primary end point was clinical/radiographic progression-free survival (PFS). The secondary end points were >50% PSA response rate and PSA progression-free survival (PSA PFS). A multivariable Cox regression model was constructed to determine whether prednisone use was independently predictive of PFS. RESULTS: We identified 200 consecutive patients for inclusion in the study: 131 men received docetaxel with prednisone and 69 received docetaxel alone. The docetaxel-prednisone cohort had superior PFS compared with the docetaxel-alone cohort (median PFS: 7.8 vs. 6.2 months, HR 0.68 (95% confidence interval (CI) 0.48-0.97), P=0.03). Prednisone use was associated with a reduced risk of progression on docetaxel in the propensity score-weighted multivariable Cox model (P=0.002). Among abiraterone- or ketoconazole-pretreated patients, no difference in PFS was observed between prednisone-containing and non-prednisone-containing docetaxel regimens (median PFS: 7.1 vs. 6.3 months, HR 0.96 (95% CI 0.59-1.57), P=0.87). CONCLUSIONS: The incorporation of prednisone potentially augments the efficacy of docetaxel in patients with mCRPC. We hypothesize that this advantage is limited to patients who have not previously received corticosteroids. Prospective confirmation is needed.

A supramicromolar elevation of intracellular free calcium ([Ca(2+)](i)) is consistently required to induce the execution phase of apoptosis.

Many agents, such as the endoplasmic reticulum Ca(2+) ATPase inhibitor, thapsigargin, or the ionophore, ionomycin, induce apoptosis by transiently elevating [Ca(2+)](i). The role of [Ca(2+)](i) in apoptosis induced by agents that do not immediately increase [Ca(2+)](i), such as 5-FdUr, TGF beta-1, doxorubicin, or radiation, is far more controversial. In the present paper, [Ca(2+)](i) was measured continuously for 120 h. in prostate and bladder cancer cell lines exposed to these four agents: 5-FdUR, TGF beta-1, doxorubicin, or radiation. Each of them consistently induced a delayed [Ca(2+)](i) rise associated with the morphological changes that characterize the execution phase of apoptosis (i.e. rounding, blebbing). This [Ca(2+)](i) rise occurred in two consecutive steps (< or = 10 microM and >10 microM) and resulted from a Ca(2+) influx from the extracellular medium. This delayed supramicromolar [Ca(2+)](i) rise was also observed previously in breast, prostate and bladder cancer cell lines exposed to thapsigargin. This influx regulated transcriptional reprogramming of Gadd153 and is required to activate cytochrome c release, caspase-3 activation, loss of clonal survival and DNA fragmentation. When cells were maintained in low extracellular Ca(2+) media, these phenomena were temporarily delayed but occurred on return to normal Ca(2+) medium. Similarly, apoptosis could be delayed by overexpressing the Ca(2+)-binding proteins, Calbindin-D(28K) and parvalbumin. As this delayed >or = 10 microM [Ca(2+)](i) elevation was observed in a number of cell lines exposed to a variety of different agents, we conclude that such elevation constitutes a key and general event of apoptosis in these malignant cells.

Androgens regulate vascular endothelial growth factor content in normal and malignant prostatic tissue.

In previous studies, we have demonstrated that androgen ablation-induced growth inhibition of androgen-responsive PC-82 and A-2 human prostate cancer xenografts involves not only direct activation of programmed (apoptotic) death of these cells but also indirect activation of this death process via a decrease in tumor angiogenesis secondary to a reduction in tumor vascular endothelial growth factor (VEGF) levels. To determine whether androgens consistently regulate angiogenesis via control of VEGF levels, an additional human (i.e., LnCaP) and two rodent (i.e., Dunning G and H) androgen-sensitive prostate cancer sublines were tested. Androgen ablation causes a decrease in the subsequent growth rate of each of these three additional prostate cancer sublines, and this growth inhibition is consistently associated with a >60% reduction in tumor VEGF levels. To examine whether androgens regulate VEGF levels not only in malignant but also in normal prostatic tissue, male rats were castrated, and the temporal changes in the VEGF content of ventral prostate tissue were determined. One week after castration, VEGF content decreased to <20% within the ventral prostate. Subsequent replacement with exogenous androgen to long-term castrated rats stimulated an 8-fold rise in ventral prostate VEGF content within 1 week. To evaluate whether androgen regulation of VEGF is due to a direct effect of androgen on prostatic cells, the dose-response ability of androgens to increase VEGF levels in media of LnCaP cells grown in vitro was tested. These studies demonstrate that androgens directly stimulate VEGF secretion in these cells. The presence of 4-5-fold higher levels of VEGF in prostatic fluid versus seminal vesicle fluid obtained from benign prostatic hyperplasia and clinically localized prostate cancer patients suggests that elevated levels of VEGF may contribute to the progression of these prostatic conditions by promoting angiogenesis. In summary, one of the mechanisms for androgen sensitivity for the control of the growth of both normal and malignant prostatic tissue is via its stimulation of VEGF levels.

Pan-trk inhibition decreases metastasis and enhances host survival in experimental models as a result of its selective induction of apoptosis of prostate cancer cells.

During the progression of prostate cancer, molecular changes occur resulting in the autocrine production of a series of neurotrophins by the malignant cells. This is coupled with expression of high-affinity cognate receptors for these ligands, termed trk receptors, by these cancer cells. The binding of the neurotrophins to their trk receptors activates the receptor's latent tyrosine kinase activity inducing a series of signal transduction pathways within these prostate cancer cells. These molecular changes result in the acquisition by prostate cancer cells of a restricted requirement for these trk signaling pathways for optimal survival. CEP-701 is an indolocarbazole compound specifically designed as a potent inhibitor (IC(50), 4 nM) of the tyrosine kinase activity of the trk receptors required for initiation of these survival pathways. In the present studies, the consequences of CEP-701 inhibition of these trk signaling survival pathways were tested in vivo using both rat (R3327 AT 6.3 and H) and human (TSU-pr1 and CWR-22Rv1) prostatic cancer models. These in vivo studies demonstrated that treatment with CEP-701 inhibits the growth of both rodent and human prostate cancers, without being toxic to the normal tissue including the host prostate. Because of this selective effect, CEP-701 inhibits metastasis and growth of both primary and metastatic sites of prostate cancer. Based upon this profile, long-term survival studies were performed using the slow-growing Dunning H rat prostate cancer model. For these latter studies, the dosing regimen was 10 mg CEP-701/kg/dose twice a day via gavage 5 days a week. This regimen maintains CEP-701 tumor tissue concentrations of 25-50 nM. Such chronic dosing increased (P < 0.001) the median survival of rats bearing the slow growing H prostate cancers from 408 days (395-432 days, 95% confidence interval) for the vehicle group (n = 18) to 566 days (497-598 days, 95% confidence interval) for the CEP-701-treated group (n = 24).

Delayed micromolar elevation in intracellular calcium precedes induction of apoptosis in thapsigargin-treated breast cancer cells.

Thapsigargin (TG), a highly specific inhibitor of the sarcoplasmic reticulum and endoplasmic reticulum Ca2+-ATPase pump, can induce apoptosis in a variety of epithelial and lymphoid cell types. In prostate cancer cell lines, TG induces an initial 5- to 10-fold elevation of intracellular calcium ([Ca2+]i) within a few minutes of exposure. With prolonged exposure times (i.e., 12-36 h) a second elevation of [Ca2+]i to >10 microM is observed. In this study, the human breast carcinoma cell lines MCF-7 and MDA MB 468 cells were used to determine the temporal relationship between TG-induced elevation of [Ca2+]i and activation of programmed cell death. Using a microinjection method that allows for long-term analysis of [Ca2+]i changes, we found that after TG exposure, calcium measurements in these cells demonstrated an initial rise (>4-fold) in [Ca2+]i that occurred within minutes and returned to baseline within a few hours. With prolonged TG exposure, the cells underwent a second elevation (>5 microM) of [Ca2+]i occurring stochastically between 12 and 36 h after the initial exposure to TG. Both of the cell lines were growth-inhibited by 100 nM TG after only 1 h of exposure, but clonogenic ability in the MCF-7 cells was significantly reduced only after 48 h of exposure. The induction of apoptosis by TG was demonstrated by morphological changes typical for programmed cell death and DNA fragmentation (both high molecular weight and oligonucleosomal-sized fragments were detected) after 48 h of treatment. TG induction of apoptosis in these breast cancer cells occurred subsequent to the secondary rise in [Ca2+]i, which confirmed that this secondary rise in [Ca2+]i is not prostate cancer-specific. The secondary rise in [Ca2+]i to micromolar levels may directly activate the endonucleases responsible for DNA fragmentation that occurs as part of the apoptotic process. These studies indicate that TG is an active agent in vitro against breast cancer cells. Inactive prodrug analogues of TG are currently being developed that can be activated by tissue-specific proteases, and further pursuit of this strategy as a potential treatment for breast cancer is warranted.

Expression of prostate-specific membrane antigen in transitional cell carcinoma of the bladder: prognostic value?

The expression of Prostate-specific membrane antigen (PSMA) mRNA was assessed in the normal bladder urothelium (n = 9), transitional cell carcinoma (TCC) specimens (n = 52), TCC-derived cell lines (n = 3), and preoperative blood samples from TCC patients (n = 27). Specific PSMA mRNA was found in 100% of normal and malignant tissues and two cell lines. PSMA protein was detected in normal (n = 3) and malignant tissues (n = 4). Using a PSMA-specific substrate, PSMA enzymatic activity was found in two bladder cell lines and correlated with immunostaining. Seven of the 27 TCC preoperative blood samples were positive by reverse transcription-PCR. These preliminary results, obtained on a nonrandomized cohort of patients, correlated with tumor invasion (positive RT-PCR: 0% for pT < or = 2 versus 41% for pT > or = 3) and 2-year survival rate (81% in the PSMA-negative group versus 29% in the PSMA-positive group). Although the clinical usefulness of this assay requires confirmation in larger prospective randomized trials, current preliminary results suggest that a blood-borne PSMA mRNA PCR assay may be a useful tool to predict a poor outcome in TCC patients.

CCAAT/Enhancer binding protein ß controls androgen-deprivation-induced senescence in prostate cancer cells.

The processes associated with transition to castration-resistant prostate cancer (PC) growth are not well understood. Cellular senescence is a stable cell cycle arrest that occurs in response to sublethal stress. It is often overcome in malignant transformation to confer a survival advantage. CCAAT/Enhancer Binding Protein (C/EBP) ß function is frequently deregulated in human malignancies and interestingly, androgen-sensitive PC cells express primarily the liver-enriched inhibitory protein isoform. We found that C/EBPß expression is negatively regulated by androgen receptor (AR) activity and that treatment of androgen-sensitive cell lines with anti-androgens increases C/EBPß mRNA and protein levels. Accordingly, we also find that C/EBPß levels are significantly elevated in primary PC samples from castration-resistant compared with therapy-naive patients. Chromatin immunoprecipitation demonstrated enhanced binding of the AR to the proximal promoter of the CEBPB gene in the presence of dihydroxytestosterone. Upon androgen deprivation, induction of C/EBPß is facilitated by active transcription as evident by increased histone 3 acetylation at the C/EBPß promoter. Also, the androgen agonist R1881 suppresses the activity of a CEBPB promoter reporter. Loss of C/EBPß expression prevents growth arrest following androgen deprivation or anti-androgen challenge. Accordingly, suppression of C/EBPß under low androgen conditions results in reduced expression of senescence-associated secretory genes, significantly decreased number of cells displaying heterochromatin foci and increased numbers of Ki67-positive cells. Ectopic expression of C/EBPß caused pronounced morphological changes, reduced PC cell growth and increased the number of senescent LNCaP cells. Lastly, we found that senescence contributes to PC cell survival under androgen deprivation, and C/EBPß-deficient cells were significantly more susceptible to killing by cytotoxic chemotherapy following androgen deprivation. Our data demonstrate that upregulation of C/EBPß is critical for complete maintenance of androgen deprivation-induced senescence and that targeting C/EBPß expression may synergize with anti-androgen or chemotherapy in eradicating PC.

Assessment and validation of a microinjection method for kinetic analysis of [Ca2+]i in individual cells undergoing apoptosis.

Normal and malignant epithelial cells are induced to undergo apoptosis by a large variety of mechanistically diverse agents. Regardless of inducing agent, apoptosis characteristically occurs asynchronously within a population of epithelial cells over a period of 12-96 h and is associated with permeability and enzymatic perturbations. Pre-loading of cells with acetoxymethyl esters (AM) derivatives of fluorescent Ca2+ indicators (i.e. fura-2, indo-1, fluo-3) by passive diffusion allows longitudinal kinetic analysis of acute [Ca2+] changes subsequent to exposure to apoptosis inducing agents. Using prostate cancer cell lines, however, it is demonstrated that dye leakage and compartmentalization into organelles limit such passive loading to longitudinal [Ca2+]i measurements of < 2 h. Post-loading of cells exposed to the apoptosis inducing agent for several hours is also inaccurate owing to decreased loading efficiency and de-esterification of the probes resulting in increased production of fluorescent Ca(2+)-insensitive dye species. To accurately measure kinetics of [Ca2+]i changes longitudinally in individual cells undergoing apoptosis, cells were microinjected with fura dextran and maintained in a physiologic environment. [Ca2+] and morphological changes characteristic of apoptosis were then followed simultaneously in individual cells over several days following exposure to the apoptosis inducing agent.

Design, synthesis, and pharmacological evaluation of thapsigargin analogues for targeting apoptosis to prostatic cancer cells.

A series of thapsigargin (TG) analogues, containing an amino acid applicable for conjugation to a peptide specifically cleaved by prostate-specific antigen (PSA), has been prepared to develop the drug-moiety of prodrugs for treatment of prostatic cancer. The analogues were synthesized by converting TG into O-8-debutanoylthapsigargin (DBTG) and esterifying O-8 of DBTG with various amino acid linkers. The compounds were evaluated for their ability to elevate the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in TSU-Pr1 cells, their ability to inhibit the rabbit skeletal muscle SERCA pump, and their ability to induce apoptosis in TSU-Pr1 human prostatic cancer cells. The activity of analogues, in which DBTG were esterified with omega-amino acids [HOOC(CH(2))(n)()NH(2), n = 5-7, 10, 11], increased with the linker length. Analogues with 3-[4-(L-leucinoylamino)phenyl]propanoyl, 6-(L-leucinoylamino)hexanoyl, and 12-(L-serinoylamino)dodecanoyl were considerably less active than TG, and analogues with 12-(L-alaninoylamino)dodecanoyl and 12-(L-phenylalaninoylamino)dodecanoyl were almost as active as TG. The 12-(L-leucinoylamino)dodecanoyl gave an analogue equipotent with TG, making this compound promising as the drug-moiety of a PSA sensitive prodrug of TG.

The effect of the frequency and duration of PSA measurement on PSA doubling time calculations in men with biochemically recurrent prostate cancer.

BACKGROUND: PSA doubling time (PSADT) is an attractive intermediate end point for assessing novel therapies in biochemically recurrent prostate cancer (BRPC). This study explores whether PSADT calculations are influenced by frequency/duration of PSA measurements, and whether statistical variability leads investigators to find false significant results. METHODS: In retrospective analyses of two BRPC cohorts: Johns Hopkins Hospital (JHH) patients who deferred therapy and placebo patients on a randomized clinical trial (RCT), we calculated changes in PSADT from early measurements to later measurements using subsets of available PSAs for patients with ≥6 and ≥9 PSAs. We simulated hypothetical single-arm trials using randomly selected, 50-patient subsets and simulated two-arm RCTs. RESULTS: JHH cohort (n=205) had median follow-up 58 months, median age 61 years and median Gleason 7. PSA variability changed with duration of PSA measurement as median within-patient PSADT increases for men with >6 PSAs ranged from 1.0 to 1.4 months by PSA subset while increases for men with ≥9 PSAs ranged from 3.9 to 4.1 months. Frequency of measurement did not change PSA variability as PSADT increase was unchanged when odd values were used instead of all values. Approximately 30% of JHH men experienced >200% increases in PSADT. Up to 62% of 50-patient single-arm simulations detected a significant PSADT change, whereas simulated RCTs did not. Results were supported in the RCT placebo cohort; 46% of patients experienced PSADT increases >200%. CONCLUSIONS: These data suggest that calculated PSADT in BRPC may naturally increase over time in the absence of therapy and may be influenced by duration of PSA follow-up. As a result, single-arm trials could show false significant increases despite the lack of active treatment of these patients. Placebo-controlled RCTs including clinical end points are recommended to screen novel agents in men with BRPC to mitigate bias because of natural PSADT variability.

Deletion of p53 in human mammary epithelial cells causes chromosomal instability and altered therapeutic response.

The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. To evaluate the biological and clinical relevance of p53 loss, human somatic cell gene targeting was used to delete the TP53 gene in the non-tumorigenic epithelial cell line, MCF-10A. In all four p53-/- clones generated, cells acquired the capability for epidermal growth factor-independent growth and were defective in appropriate downstream signaling and cell cycle checkpoints in response to DNA damage. Interestingly, p53 loss induced chromosomal instability leading to features of transformation and the selection of clones with varying phenotypes. For example, p53-deficient clones were heterogeneous in their capacity for anchorage-independent growth and invasion. In addition, and of clinical importance, the cohort of p53-null clones showed sensitivity to chemotherapeutic interventions that varied depending not only on the type of chemotherapeutic agent, but also on the treatment schedule. In conclusion, deletion of the TP53 gene from MCF-10A cells eliminated p53 functions, as well as produced p53-/- clones with varying phenotypes possibly stemming from the distinct chromosomal changes observed. Such a model system will be useful to further understand the cancer-specific phenotypic changes that accompany p53 loss, as well as help to provide future treatment strategies for human malignancies that harbor aberrant p53.