Prostate Cancer Cell Lines C4-2 Research Articles

EB1089 inhibits the parathyroid hormone–related protein–enhanced bone metastasis and xenograft growth of human prostate cancer cells

Parathyroid hormone-related protein (PTHrP) plays a major role in prostate carcinoma progression and bone metastasis. Once prostate cancers become androgen-independent, treatment options become limited. Vitamin D analogues represent a potentially valuable class of agents in this clinical context. Using the prostate cancer cell line C4-2 as a model, we studied the effects of PTHrP and the noncalcemic vitamin D analogue EB1089 on markers of prostate cancer cell progression in vitro and in vivo. C4-2 is a second-generation androgen-independent LNCaP subline that metastasizes to the lymph nodes and bone when injected into nude mice and produces mixed lytic/blastic lesions, mimicking the in vivo situation. We report that PTHrP increases cell migration and invasion, and that a pathway via which EB1089 inhibits these processes is through down-regulation of PTHrP expression. PTHrP also increases anchorage-independent cell growth in vitro and xenograft growth in vivo; EB1089 reverses these effects. The in vivo PTHrP effects are accompanied by increased tumor cell proliferation and survival. Treatment with EB1089 reverses the proliferative but not the antiapoptotic effects of PTHrP. PTHrP also increases intratumor vessel density and vascular endothelial growth factor expression; EB1089 reverses these effects. Intracardially injected C4-2 cells produce predominantly osteoblastic lesions; PTHrP overexpression decreases the latency, increases the severity and alters the bone lesion profile to predominantly osteolytic. EB1089 largely reverses these PTHrP effects. A direct correlation between PTHrP immunoreactivity and increasing tumor grade is observed in human prostate cancer specimens. Thus, decreasing PTHrP production by treatment with vitamin D analogues may prove therapeutically beneficial for prostate cancer.

Anti‐PSMA immunotoxin as novel treatment for prostate cancer? High and specific antitumor activity on human prostate xenograft tumors in SCID mice

Expression of the prostate specific membrane antigen (PSMA) is highly restricted to prostate epithelial cells. Therefore, toxin-based immunotherapy against this antigen may represent an alternative therapeutic option for prostate cancer. For these purposes, the effects of the recombinant anti-PSMA immunotoxin A5-PE40 on prostate tumor growth were investigated in vitro and in vivo. The in vitro binding and cytotoxicity of A5-PE40 were tested on the PSMA-expressing prostate cancer cell line C4-2 and on the PSMA-negative cell line DU145 by flow cytometry and WST assays. The binding of the immunotoxin to SCID mouse xenografts and to various mouse organs was examined by Western blot analysis. In vivo, the antitumor activity of the immunotoxin was tested by injecting A5-PE40 in mice bearing C4-2 or DU145 xenografts. In vitro, a specific binding of A5-PE40 to C4-2 cells could be shown with a concentration-dependent cytotoxicity (IC(50) value=220 pM). In the next step, a specific binding of the immunotoxin to C4-2 xenografts could be demonstrated. In contrast, no binding on mouse organs expressing high homologous mouse PSMA was found. The treatment of mice with C4-2 tumors caused a significant inhibition of tumor growth in vivo, whereas DU145 xenografts remained totally unaffected. A5-PE40 represents a recombinant anti-PSMA immunotoxin with potent antitumor activity in mice bearing human prostate cancer xenograft tumors. Therefore, A5-PE40 could be a promising candidate for therapeutic applications in patients with prostate cancer.

PTHrP contributes to the anti-proliferative and integrin α6β4-regulating effects of 1,25-dihydroxyvitamin D 3

Parathyroid hormone-related protein (PTHrP) increases the growth and metastatic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D 3 [1,25(OH) 2D 3] suppresses PTHrP expression and exerts an anti-proliferative effect in prostate carcinoma cells. We used the human prostate cancer cell line C4-2 as a model system to ask whether down-regulation of PTHrP expression by 1,25(OH) 2D 3 plays a role in the anti-proliferative effects of 1,25(OH) 2D 3. Since PTHrP increases the expression of the pro-invasive integrin α6β4, we also asked whether 1,25(OH) 2D 3 decreases integrin α6β4 expression in C4-2 cells, and whether modulation of PTHrP expression by 1,25(OH) 2D 3 plays a role in the effects of 1,25(OH) 2D 3 on integrin α6β4 expression. Two strategies were utilized to modulate PTHrP levels: overexpression of PTHrP (−36 to +139) and suppression of endogenous PTHrP expression using siRNAs. We report a direct correlation between PTHrP expression, C4-2 cell proliferation and integrin α6β4 expression at the mRNA and cell surface protein level. Treatment of parental C4-2 cells with 1,25(OH) 2D 3 decreased cell proliferation and integrin α6 and β4 expression. These 1,25(OH) 2D 3 effects were significantly attenuated in cells with suppressed PTHrP expression. 1,25(OH) 2D 3 regulates PTHrP expression via a negative vitamin D response element (nVDRE) within the noncoding region of the PTHrP gene. The effects of 1,25(OH) 2D 3 on cell proliferation and integrin α6β4 expression were significantly attenuated in cells overexpressing PTHrP (−36 to +139), which lacks the nVDRE. These findings suggest that one of the pathways via which 1,25(OH) 2D 3 exerts its anti-proliferative effects is through down-regulation of PTHrP expression.

Epinephrine Protects Cancer Cells from Apoptosis via Activation of cAMP-dependent Protein Kinase and BAD Phosphorylation

The stress hormone epinephrine is known to elicit multiple systemic effects that include changes in cardiovascular parameters and immune responses. However, information about its direct action on cancer cells is limited. Here we provide evidence that epinephrine reduces sensitivity of cancer cells to apoptosis through interaction with beta(2)-adrenergic receptors. The antiapoptotic mechanism of epinephrine primarily involves phosphorylation and inactivation of the proapoptotic protein BAD by cAMP-dependent protein kinase. Moreover, BAD phosphorylation was observed at epinephrine concentrations found after acute and chronic psychosocial stress. Antiapoptotic signaling by epinephrine could be one of the mechanisms by which stress promotes tumorigenesis and decreases the efficacy of anti-cancer therapies.

Antitumor effects of etodolac, a selective cyclooxygenase-II inhibitor, against human prostate cancer cell lines in vitro and in vivo

ObjectivesTo investigate the effects of the selective cyclooxygenase-2 (COX-2) inhibitor etodolac on prostate cancer cell lines in vitro and in vivo and on E-cadherin expression in prostate cancer cells. MethodsWe evaluated the cytotoxicity of etodolac on the three prostate cancer cell lines LNCaP, C4-2, and PC-3. We also performed quantitative real-time polymerase chain reaction to measure the mRNA expression of COX-2, Bcl-2, and E-cadherin in these cell lines after etodolac treatment. In addition, we investigated the in vivo antitumor effects of etodolac on a human prostate cancer xenograft model. ResultsEtodolac exhibited significant antitumor effect in vivo and in vitro. The cytotoxicity of etodolac in LNCaP and C4-2 was markedly increased at a dose of 1000 nM in a time-dependent and dose-dependent manner. In the in vivo tumor growth study, the etodolac-treated mice exhibited more significant cytotoxicity than the phosphate-buffered saline-treated mice. Expression of E-cadherin after etodolac treatment tended to increase and that of Bcl-2 to decrease, but the expression of COX-2 had no definite tendency. ConclusionsThe COX-2 inhibitor etodolac exhibited an antitumor effect on prostate cancer cell lines in vitro and in vivo, and it might be useful for the treatment of hormone-resistant prostate cancer.

Role of SRC-1 in the Promotion of Prostate Cancer Cell Growth and Tumor Progression

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor-negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.

Molecular targeting of prostate cancer with cytotoxins and radioisotopes linked to fully human monoclonal antibodies against prostate-specific membrane antigen

2560 Background: Prostate-specific membrane antigen (PSMA) is an attractive target for immunotherapy of prostate cancer due to its abundant expression on prostate cancer cells and its up-regulation on metastatic disease. The objectives of this study were to evaluate the ability of fully human anti-PSMA monoclonal antibodies (mAbs) to specifically deliver cytotoxic payloads to prostate cancer cells in vitro and in vivo. Methods: Using novel recombinant forms of PSMA and the XenoMouse™ technology, we previously generated a panel of fully human mAbs against PSMA. Characterization of these antibodies was conducted using Biacore, ELISA, flow cytometry, anti-PSMA Western blot and other functional assays. In this study, the therapeutic potential of radiolabeled mAbs and immunotoxins was comparatively evaluated in cell-based in vitro cytotoxicity assays and in vivo xenografted animals. Results: We demonstrated that these fully human mAbs specifically recognize conformational, dimer-specific epitopes on PSMA with subnanomolar affinity. The mAbs were modified with bifunctional chelates for radiolabeling or conjugated with potent cytotoxins. The binding of the mAbs to cell-surface PSMA was virtually unchanged following conjugation. The conjugated mAbs were internalized rapidly upon binding to PSMA on tumor cells (∼60% in 2 hours) which provides efficient delivery of cytotoxins. Immunotoxins killed PSMA-expressing tumor cells with pM potency and ∼1000-fold selectivity. In tumor-bearing animal models, mAbs radiolabeled with Lu-177, a beta-emitting isotope, selectively targeted PSMA-expressing tumors, with a high tumor uptake of 30% injected dose per gram of tumor. In an aggressive tumor model using 3T3-PSMA cells, a single 400 μCi dose of Lu-177 labeled antibody significantly delayed tumor growth and increased median survival time by 3.5-fold compared to untreated control animals (p<0.005). Additional xenograft studies have been initiated using the androgen-independent human prostate cancer cell line C4-2. Conclusions: Fully human PSMA mAbs can selectively deliver cytotoxic payloads to control prostate tumor growth in vitro and in vivo. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration PSMA Development Company, LLC

Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen-producing tumors in athymic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines.

LNCaP lineage-derived human prostate cancer cell lines C4-2 and C4-2B4 acquire androgen independence and osseous metastatic potential in vivo. Using C4-2 and C4-2B4 the goals of the current investigation were 1) to establish an ideal bone xenograft model for prostate cancer cells in intact athymic or SCID/bg mice using an intraosseous route of tumor cell administration and 2) to compare prostate cancer metastasis by administering cells either through intravenous (i.v.) or intracardiac administration in athymic or SCID/bg mice. Subsequent to tumor cell administration, prostate cancer growth in the skeleton was assessed by radiographic bone density, serum prostate-specific antigen (PSA) levels, presence of hematogenous prostate cancer cells and histopathologic evaluation of tumor specimens in the lymph node and skeleton. Our results show that whereas LNCaP cells injected intracardially failed to develop metastasis, C4-2 cells injected similarly had the highest metastatic capability in SCID/bg mice. Retroperitoneal and mediastinal lymph node metastases were noted in 3/7 animals, whereas 2/7 animals developed osteoblastic spine metastases. Intracardiac injection of C4-2 in athymic hosts produced spinal metastases in 1/5 animals at 8-12 weeks post-injection; PC-3 injected intracardially also metastasized to the bone but yielded osteolytic responses. Intravenous injection of either LNCaP or C4-2 failed to establish tumor colonies. Intrailiac injection of C4-2 but not LNCaP nor C4-2B4 cells in athymic mice established rapidly growing tumors in 4/8 animals at 2-7 weeks after inoculation. Intrafemoral injection of C4-2 (9/16) and C4-2B4 (5/18) but not LNCaP (0/13) cells resulted in the development of osteoblastic bone lesions in athymic mice (mean: 6 weeks, range: 3-12 weeks). In SCID/bg mice, intrafemoral injection of LNCaP (6/8), C4-2 (8/8) and C4-2B4 (8/8) cells formed PSA-producing, osteoblastic tumors in the bone marrow space within 3-5 weeks after tumor cell inoculation. A stepwise increase of serum PSA was detected in all animals. Reverse transcription-polymerase chain reaction (RT-PCR) to detect hematogenously disseminated prostate cancer cells could not be correlated to either serum PSA level or histological evidence of tumor cells in the marrow space. We have thus established a PSA-producing and osteoblastic human prostate cancer xenograft model in mice.

Suramin-induced decrease in prostate-specific antigen expression with no effect on tumor growth in the LNCaP model of human prostate cancer.

Suramin, a polysulfonated naphthylurea and a recognized antitrypanosomal agent, has shown some promise in phase II clinical trials in the management of hormone-refractory human prostate cancer. Reduction of serum prostate-specific antigen (PSA) levels has been proposed as an end point for evaluating the antitumor efficacy of treatments for hormone-refractory prostate cancer. We examined the antitumor effect of suramin in an in vivo mouse model of hormone-refractory human prostate cancer to determine whether a decrease in PSA levels reflects a reduction in tumor growth (volume). The tumors were induced in castrated, athymic nude mice by use of the androgen-independent, tumorigenic human prostate cancer cell line C4-2, which is a subline of the androgen-dependent, parental nontumorigenic cell line LNCaP. We also evaluated the effects of suramin in vitro on cell growth and the expression of PSA messenger RNA (mRNA) in both LNCaP and C4-2 cells. For the in vivo studies, 24 mice were given a subcutaneous injection of 5 x 10(6) C4-2 cells at each of four sites. Animals (n = 20) with tumor volumes greater than 1 mm3 or less than 5 mm3 were divided equally into two groups. Drug treatment was initiated in one group by administration of 1 mg suramin intraperitoneally, followed by 0.1 mg suramin at 10-day intervals to maintain constant serum levels. Tumor growth and PSA expression levels were monitored. For the in vitro studies, both LNCaP and C4-2 cells were exposed to 100-400 microgram/mL suramin, and cell growth was monitored by a quantitative crystal violet assay. PSA mRNA expression was assessed by northern blot analysis in cells treated with either 250 microgram/mL suramin, 400 ng/mL dihydrotestosterone (DHT) (positive control), or 0.5-75 microgram/mL hydrocortisone (to mimic the clinical use of hydrocortisone during suramin treatment to compensate for the loss of adrenocortical function). In some studies, the combined effect of DHT and suramin on PSA mRNA expression was also evaluated. A two-way analysis of variance was performed to evaluate the treatment differences, and P values were obtained from two-sided tests for statistical significance. In vivo, suramin did not significantly affect the growth of androgen-independent C4-2 tumors (relative to the growth of tumors in 5% glucose-treated control animals; P = .76). However, suramin significantly decreased the ratio of PSA level to tumor volume (ng/mL PSA per mm(3) of tumor) (P<.001). Mice developed bone metastases in both treatment arms. Suramin affected the in vitro growth of LNCaP cells but not of C4-2 cells. Suramin diminished PSA mRNA expression in both LNCaP and C4-2 cells grown in vitro. Hydrocortisone had no effect on PSA mRNA levels. Although suramin inhibited the growth of androgen-dependent LNCaP cells, it did not inhibit the growth of androgen-independent C4-2 cells either in vitro or in vivo. Suramin significantly decreased PSA mRNA expression in both cell lines in vitro and depressed serum PSA levels in mice bearing androgen-independent C4-2 tumors. PSA level should be used with caution as an end point in clinical trials using suramin therapy for hormone-refractory prostate cancer.