PC3 Metastatic Prostate Cancer Cells Research Articles

Protein Kinase D3 (PKD3) Requires Hsp90 for Stability and Promotion of Prostate Cancer Cell Migration.

Prostate cancer metastasis is a significant cause of mortality in men. PKD3 facilitates tumor growth and metastasis, however, its regulation is largely unclear. The Hsp90 chaperone stabilizes an array of signaling client proteins, thus is an enabler of the malignant phenotype. Here, using different prostate cancer cell lines, we report that Hsp90 ensures PKD3 conformational stability and function to promote cancer cell migration. We found that pharmacological inhibition of either PKDs or Hsp90 dose-dependently abrogated the migration of DU145 and PC3 metastatic prostate cancer cells. Hsp90 inhibition by ganetespib caused a dose-dependent depletion of PKD2, PKD3, and Akt, which are all involved in metastasis formation. Proximity ligation assay and immunoprecipitation experiments demonstrated a physical interaction between Hsp90 and PKD3. Inhibition of the chaperone-client interaction induced misfolding and proteasomal degradation of PKD3. PKD3 siRNA combined with ganetespib treatment demonstrated a specific involvement of PKD3 in DU145 and PC3 cell migration, which was entirely dependent on Hsp90. Finally, ectopic expression of PKD3 enhanced migration of non-metastatic LNCaP cells in an Hsp90-dependent manner. Altogether, our findings identify PKD3 as an Hsp90 client and uncover a potential mechanism of Hsp90 in prostate cancer metastasis. The molecular interaction revealed here may regulate other biological and pathological functions.

Curcumin and Carnosic Acid Cooperate to Inhibit Proliferation and Alter Mitochondrial Function of Metastatic Prostate Cancer Cells.

Anticancer activities of plant polyphenols have been demonstrated in various models of neoplasia. However, evidence obtained in numerous in vitro studies indicates that proliferation arrest and/or killing of cancer cells require quite high micromolar concentrations of polyphenols that are difficult to reach in vivo and can also be (geno)toxic to at least some types of normal cells. The ability of certain polyphenols to synergize with one another at low concentrations can be used as a promising strategy to effectively treat human malignancies. We have recently reported that curcumin and carnosic acid applied at non-cytotoxic concentrations synergistically cooperate to induce massive apoptosis in acute myeloid leukemia cells, but not in normal hematopoietic and non-hematopoietic cells, via sustained cytosolic calcium overload. Here, we show that the two polyphenols can also synergistically suppress the growth of DU145 and PC-3 metastatic prostate cancer cell cultures. However, instead of cell killing, the combined treatment induced a marked inhibition of cell proliferation associated with G0/G1 cell cycle arrest. This was preceded by transient elevation of cytosolic calcium levels and prolonged dissipation of the mitochondrial membrane potential, without generating oxidative stress, and was associated with defective oxidative phosphorylation encompassing mitochondrial dysfunction. The above effects were concomitant with a significant downregulation of mRNA and protein expression of the oncogenic kinase SGK1, the mitochondria-hosted mTOR component. In addition, a moderate decrease in SGK1 phosphorylation at Ser422 was observed in polyphenol-treated cells. The mTOR inhibitor rapamycin produced a similar reduction in SGK1 mRNA and protein levels as well as phosphorylation. Collectively, our findings suggest that the combination of curcumin and carnosic acid at potentially bioavailable concentrations may effectively target different types of cancer cells by distinct modes of action. This and similar combinations merit further exploration as an anticancer modality.

Etoposide Loaded SPION-PNIPAM Nanoparticles Improve the in vitro Therapeutic Outcome on Metastatic Prostate Cancer Cells via Enhanced Apoptosis.

Prostate cancer is among the leading causes of death worldwide because its metastatic form is a deadly disease. Therefore, the development of new chemotherapeutics is of immense importance. Nanoparticle technology seems to provide diverse options in this regard. Therefore, poly(N-isopropylacrylamide) (PNIPAM) coated superparamagnetic iron oxide nanoparticles (SPION) loaded with Etoposide were prepared in small sizes (57 nm) and with 3.5 % drug content to improve the efficiency of Etoposide in prostate cancer therapy. Sustained release of the drug was achieved, which found to be sensitive to low pH and high temperature. The anti-growth activity of SPION-PNIPAM-Etoposide formulation against metastatic prostate cancer cells (PC-3, LNCaP) were investigated by SRB assay, then, confirmed by ATP assay. Mode of cell death was evaluated by using flow cytometry analyses. A significant improvement of nanoformulated drug was observed at 5-10 μg/ml doses of the drug in both cell lines. More importantly, this formulation enhanced the cytotoxic effect of Etoposide on PC-3 cells, which is considered more resistant to Etoposide than LNCaP and reduced the IC50 value by 55 % reaching to 4.5 μg drug/ml, which is a very significant improvement in the literature. It was clearly shown that nanoformulated drug provided about 3-fold increases in caspase-dependent early apoptotic cells in PC-3 cells. The novel formulation seems to successfully cause cell death of especially PC-3 metastatic prostate cancer cells. It should therefore be taken into consideration for further animal studies as a novel potent anticancer agent.

Eprinomectin, a novel semi-synthetic macrocylic lactone is cytotoxic to PC3 metastatic prostate cancer cells via inducing apoptosis

Prostate Cancer (PCa) is the second most common cancer among men in United States after skin cancer. Conventional chemotherapeutic drugs available for PCa treatment are limited due to toxicity and resistance issues. Therefore, there is an urgent need to develop more effective treatment for advanced PCa. In this current study, we focused on evaluating the anti-cancer efficacy of Eprinomectin (EP), a novel avermectin analog against PC3 metastatic PCa cells. EP displayed robust inhibition of cell viability of PC3 cells in addition to suppressing the colony formation and wound healing capabilities. Our study showed that EP targets PC3 cells via inducing ROS and apoptosis activation. EP treatment enforces cell cycle arrest at G0/G1 phase via targeting cyclin-dependent kinase 4 (CDK4) and subsequent induction of apoptosis in PC3 cells. At the molecular level, EP effectively inhibited the expression of various cancer stem cell markers such as ALDH1, Sox-2, Nanog, Oct3/4 and CD44. Interestingly, EP also inhibited the activity of alkaline phosphatase, a maker of pluripotent stem cells. Of note, EP treatment resulted in the translocation of β-catenin from the nucleus to the cytoplasm indicating that EP antagonizes Wnt/β-catenin signaling pathway. Western blotting analysis revealed that EP downregulated the expression of key cell cycle markers such as cyclin D1, cyclin D3, CDK4, and c-Myc. In addition, EP inhibited the anti-apoptotic markers such as Mcl-1, XIAP, c-IAP1 and survivin in PC3 cells. On the other hand, EP treatment resulted in the activation of pH2A.X, Bad, caspase-9, caspase-3 and cleavage of PARP1. Taken together, our data suggests that EP is a potential agent to treat advanced PCa cells via modulating apoptosis signaling.

A New Strategy for the Synthesis of Nonsymmetrical 3,3’-(Aryl/alkyl- methylene) bis-2-hydroxy-1,4-naphthoquinones and Their Cytotoxic Effects in PC3 Prostate Cancer Cells

A novel method for the synthesis of nonsymmetrical 3,3’-(aryl/alkyl-methylene)bis-2-hydroxy-1,4-naphthoquinones was developed by using the Mannich adduct of naphthoquinone and the reaction with another moiety of 2-hydroxy-1,4-naphthoquinone. This novel method produces for the first time nonsymmetrical 3,3’-(aryl/alkyl-methylene)bis-2-hydroxy-1,4-naphthoquinones. In a preliminary study, these compounds (15c, 15f and 15h) were evaluated regarding their effect over the viability of PC3 metastatic prostate cancer cell using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays at 100 µM. Three of these compounds presented relevant cytotoxic effects at 72 h posttreatment.

Nitro aspirin (NCX4040) induces apoptosis in PC3 metastatic prostate cancer cells via hydrogen peroxide (H2O2)-mediated oxidative stress

Non-steroidal anti-inflammatory drugs (NSAID) have shown promise as anticancer agents by inducing cell death apart from their antipyretic, anti-inflammatory and anti-thrombogenic effects. In our current study, we investigated the oxidative stress mediated cell death mechanism of a NSAID derivative NCX4040 (a nitric oxide (NO) releasing form of aspirin) in castration-resistant prostate cancer (CRPC) PC3 cell line. Our data revealed that NCX4040 is more potent than its parent compound aspirin or NO releasing compound DETA NONOate. NCX4040 significantly induced hydrogen peroxide formation with ensuing oxidative stress and mitochondrial depolarization resulting in lipid peroxidation, cell cycle arrest, inhibition of colony growth and induction of apoptosis in PC3 cells. Moreover, NCX4040 inhibited migration potential of PC3 cells by depolymerizing F-actin and promoting anoikis. Interestingly, elevated levels of NADPH oxidase 1 (NOX1), superoxide dismutase (SOD) 1 and 2 were observed upon NCX4040 treatment. However, down regulation of anti-apoptotic markers B-cell lymphoma 2 (Bcl2) and anti-oxidant thioredoxin reductase 1 (TXNRD1) expression were observed. In addition, NCX4040 down regulated cyclin D1 expression in PC3 cells further supporting the anticancer effect of NCX4040. Western blot analysis revealed that significant down regulation of key anti-apoptotic markers such as cellular inhibitor of apoptosis protein-1 (cIAP1), X-linked inhibitor of apoptosis (XIAP), survivin, and Cellular-Myc (c-Myc). On the other hand, NCX4040-treated cells showed upregulation of phosho histone H2AX (pH2AX), cleaved caspase3 and cleaved Poly [ADP-ribose] polymerase 1 (PARP1). Taken together, our data demonstrate that NCX4040 treatment enhances free radical formation which in turn induces oxidative stress leading to mitochondrial mediated cell death in metastatic PC3 cells.

Abstract 5171: GRP78 as a potential modulator of cell adhesion markers in metastatic prostate cancer and multiple myeloma

Abstract Background: Glucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment. Methods: N-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells. Results: We demonstrate that GRP78 KD: 1) induced the concomitant downregulation of N-cad in both PC3 and MM.1S cells, 2) resulted in significant decreases of both N-cad and E-cad protein levels in PC3 cells, 3) induced TGF-β1 and Snail-2 expression, potentially accounting for the observed downregulation of E-cad. Our data also suggests that N-cad regulation via GRP78 KD supersedes the effects of TGF-β1; i.e., N-cad was downregulated in spite of the fact that TGF-β1 expression was significantly increased upon GRP78 KD. Conclusion: This work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche. Citation Format: Christopher Cultrara, David Sabatino, Jenny Zilberberg. GRP78 as a potential modulator of cell adhesion markers in metastatic prostate cancer and multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5171.

GRP78 modulates cell adhesion markers in prostate Cancer and multiple myeloma cell lines

BackgroundGlucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment.MethodsN-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells.ResultsGRP78 KD led to concomitant downregulation of N-cad in both tumors types. In PC3 cells, GRP78 KD significantly decreased E-cadherin (E-cad) expression likely associated with the induction in TGF-β1 expression. Furthermore, GRP78 KD also triggered drastic changes in PC3 cells morphology and decreased their adhesion to osteoblasts (OSB) dependent, in part, to the reduced N-cad expression.ConclusionThis work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche.

Abstract 1090: The role of connexin43 in prostate cancer motility

Abstract Prostate cancer (PC) is one of the most common male cancers in United States. PC lethality is due to metastatic growth, when cancer cells move from the primary tumor site to a secondary site. A key step in metastsis is the acquisition of motility. Connexins (Cx) play a major role in cell-cell communication and growing evidence demonstrates their contribution to cell motility. Cx43 is up-regulated in cultured PC3 metastatic prostate cancer cells. To understand the role of Cx43 in migration we asked whether silencing Cx43 would decrease PC3 cell migration. Using cells in which short interfering RNA (sh-RNA) silenced Cx43 mRNA, we showed that silencing Cx43 decreased motility of sh-Cx43 PC3 cells in Boyden chamber assays. To confirm these results in another biological system, we performed cellular wound healing assays. Silencing Cx43 decreased motility of PC3 cells reducing movement to wounded area, leaving more area open in the Cx43 inhibited cell lines. The wound healing assay showed that knockdown Cx43 altered directional movement, velocity and distance. These results supported those of our Boyden assays showing that loss of Cx43 decreases the migratory ability of PC3. Conversely, the overexpression of Cx43 in LNCaP cells, non-migratory prostate cancer cells that lack Cx43 expression, increased their motility. A comparison of cell lines engineered to overexpress or to silence Cx43 confirms the importance of Cx43 in the motility of prostate cancer cells in vitro. Although the mechanism whereby Cx43 regulates motility is still unclear, our preliminary data revealed that Cx43 co-immunoprecipitated with N-cadherin, suggesting that they form a complex in PC3 cells. Cell fractionation studies of PC3 control cells showed that N-cadherin was located in the cell membrane and not detected free in the cytoplasm, but in sh-Cx43 PC3 cells that lack Cx43, the distribution of N-cadherin protein was altered and significant levels of N-cadherin were also detected in cytoplasmic fractions. Whether changes in N-cadherin distribution is a direct or indirect result of loss of Cx43 in sh-Cx43 PC3 cells, is unclear. However, modulation of Cx43 levels in prostate cancer cells does alter motility and N-cadherin distribution. These results indicate that Cx43 and N-cadherin are in close association in PC3 cells and their interaction may play an important role in prostate cancer cell motility. A better understanding of the requirements of migratory PC cells may lead to more effective targeting of key steps in PC progression to lethal metastatic disease. Citation Format: Abdulaziz A. Aloliqi. The role of connexin43 in prostate cancer motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1090.

Abstract 4483: TGF-β downregulates osteopontin isoforms and induce and epithelial plasticity in PC3 metastatic prostate cancer cells

Abstract Total osteopontin (OPN) has been described as a master regulator of epithelial-mesenchymal transition (EMT), including in prostate cancer (PCa). However, the contribution of each OPN splicing isoform (OPN-SI), named OPNa, OPNb, and OPNc in the epithelial plasticity dynamic process is currently unknown. Besides, the contribution of alternative splicing regulation in this scenario is poorly understood. Here we aimed to investigate the expression of OPN-SI and the associated splicing pattern regulation in response to metastatic prostate cancer (PCa) cells treatment with TGF-β . Further, we aimed to investigate the resulting cell phenotypic features. To achieve this, we treated PC3 cells with TGF-β 10ng/ml for 48 h as a model to induce epithelial plasticity in these cells. Total RNA was extracted and the corresponding cDNA was tested for transcriptional levels of OPN-SI, SR and HnRNP splicing regulators as well as classical (EMT) markers by using real time PCR. EMT markers and cell morphology have also been analyzed by immunofluoresce. PC3 cell phenotype has also been evaluated by using cell migration and viabilitity assays in response to β-lapachone cytotoxic reagent. PC3 cells treated with TGF-β displayed an intermediate EMT phenotype, as evidenced by a significant downregulation of both epithelial (E-cadherin, claudin-3 andcytokeratin 18) and mesenchymal markers (vimentin, N-cadherin, snailandslug). These cells presented a higher cytoplasmic E-cadherin expression, as well as a lower vimentin cytoplasmic staining. A major cortical distribution of actin filaments beneath the cell cortex has been observed. Further, a significant downregulation of OPNa and mainly, OPNb and OPNc isoforms, as well as most tested splicing regulators has been shown. TGF-β treatment also promoted a significant inhibition on cell migration as well as higher sensitivity to β-lapachone treatment, as compared to control cells. Altogether, our data evidence that in our in vitro experimental model, TGF-β promoted a an intermediate EMT phenotype, with a predominance of epithelial features, associated to downregulation of OPN-SI and most tested splicing regulators. We then hypothesize that TGF-β may also be an EMT-like inducer, in which metastatic PCa cells could acquire major epithelial features that facilitate cell anchorage and adhesion in distant tumor sites. OPN-SI dowregulation in this process could favour epithelial-like features, as apposed to OPN-SI overexpression during tumor progression towards metastic circulating cells typically displaying mesechymal phenotypes. Citation Format: Durval Marques, Rodrigo A. Peres, Paula P. de Freitas, Abigail C. Resende, Nataly dos Santos Melo, Waldemir Fernandes de Souza, José A. Morgado-Diaz, Etel Rodrigues Pereira Gimba. TGF-β downregulates osteopontin isoforms and induce and epithelial plasticity in PC3 metastatic prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2017-4483

Development of TMTP-1 targeted designer biopolymers for gene delivery to prostate cancer

Designer biopolymers (DBPs) represent state of the art genetically engineered biomacromolecules designed to condense plasmid DNA, and overcome intra- and extra- cellular barriers to gene delivery. Three DBPs were synthesized, each with the tumor molecular targeting peptide-1 (TMTP-1) motif to specifically target metastases. Each DBP was complexed with a pEGFP-N1 reporter plasmid to permit physiochemical and biological assay analysis. Results indicated that two of the biopolymers (RMHT and RM3GT) effectively condensed pEGFP-N1 into cationic nanoparticles <100nm and were capable of transfecting PC-3 metastatic prostate cancer cells. Conversely the anionic RMGT DBP nanoparticles could not transfect PC-3 cells. RMHT and RM3GT nanoparticles were stable in the presence of serum and protected the cargo from degradation. Additionally it was concluded that cell viability could recover post-transfection with these DBPs, which were less toxic than the commercially available transfection reagent Lipofectamine® 2000. With both DBPs, a higher transfection efficacy was observed in PC-3 cells than in the moderately metastatic, DU145, and normal, PNT2-C2, cell lines. Blocking of the TMTP-1 receptors inhibited gene transfer indicating internalization via this receptor. In conclusion RMHT and RM3GT are fully functional DBPs that address major obstacles to gene delivery and target metastatic cells expressing the TMTP-1 receptor.

Combined effect of sodium selenite and docetaxel on PC3 metastatic prostate cancer cell line

Docetaxel and sodium selenite are well known for their anticancer properties. While resistance to docetaxel remains an obstacle in prostate cancer chemotherapy, sodium selenite, has been exploited as a new therapeutic approach. Currently, development of therapies affecting a multitude of cell targets, have been proposed as a strategy to overcome drug resistance. This association may reduce systemic toxicity counteracting a wide range of side effects.Here we report the effect of docetaxel and sodium selenite combination on the PC3 prostate cancer cell line, derived from bone metastasis. Therefore we evaluate cell growth, cell cycle progression, viability, mitochondria membrane potential, cytochrome C, Bax/Bcl2 ratio, caspase-3 expression and reactive oxygen species production.Our results suggest that sodium selenite and docetaxel combination have a synergistic effect on cell growth inhibition (67%) compared with docetaxel (22%) and sodium selenite (24%) alone. This combination also significantly induced cell death, mainly by late apoptosis vs necrosis, which is correlated with mitochondria membrane potential depletion. On the other hand, cytochrome C, Bax/Bcl2 ratio and caspase-3, known as proapoptotic factors, significantly increased in the presence of sodium selenite alone, but not in the presence of docetaxel in monotherapy or in combination with sodium selenite.These findings suggest that docetaxel and sodium selenite combination may be more effective on prostate cancer treatment than docetaxel alone warranting further evaluation of this combination in prostate cancer therapeutic approach.

The Palmitoylation of Metastasis Suppressor KAI1/CD82 Is Important for Its Motility- and Invasiveness-Inhibitory Activity

The cancer metastasis suppressor protein KAI1/CD82 is a member of the tetraspanin superfamily. Recent studies have demonstrated that tetraspanins are palmitoylated and that palmitoylation contributes to the organization of tetraspanin webs or tetraspanin-enriched microdomains. However, the effect of palmitoylation on tetraspanin-mediated cellular functions remains obscure. In this study, we found that tetraspanin KAI1/CD82 was palmitoylated when expressed in PC3 metastatic prostate cancer cells and that palmitoylation involved all of the cytoplasmic cysteine residues proximal to the plasma membrane. Notably, the palmitoylation-deficient KAI1/CD82 mutant largely reversed the wild-type KAI1/CD82's inhibitory effects on migration and invasion of PC3 cells. Also, palmitoylation regulates the subcellular distribution of KAI1/CD82 and its association with other tetraspanins, suggesting that the localized interaction of KAI1/CD82 with tetraspanin webs or tetraspanin-enriched microdomains is important for KAI1/CD82's motility-inhibitory activity. Moreover, we found that KAI1/CD82 palmitoylation affected motility-related subcellular events such as lamellipodia formation and actin cytoskeleton organization and that the alteration of these processes likely contributes to KAI1/CD82's inhibition of motility. Finally, the reversal of cell motility seen in the palmitoylation-deficient KAI1/CD82 mutant correlates with regaining of p130(CAS)-CrkII coupling, a signaling step important for KAI1/CD82's activity. Taken together, our results indicate that palmitoylation is crucial for the functional integrity of tetraspanin KAI1/CD82 during the suppression of cancer cell migration and invasion.

213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model

213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model

213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model

213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model

213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model

A novel α-particle emitting (213Bi) plasminogen activator inhibitor type 2 construct, which targets the membrane-bound urokinase plasminogen activator on prostate cancer cells, was prepared and evaluated in vitro and in a xenograft animal model. The PC3 prostate cancer cell line expresses urokinase plasminogen activator which binds to its receptor on the cell membrane; plasminogen activator inhibitor type 2 is bound to urokinase plasminogen activator/urokinase plasminogen activator receptor to form stable complexes. In vitro, the cytotoxicity of 213Bi-plasminogen activator inhibitor type 2 against prostate cancer cells was tested using the MTS assay and apoptosis was documented using terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) assay. In vivo, antiproliferative effects for tumours and prostate cancer lymph node metastasis were carried out in an athymic nude mouse model with a subcutaneous xenograft of PC3 cells. 213Bi-plasminogen activator inhibitor type 2 was specifically cytotoxic to PC3 cells in a concentration-dependent fashion, causing the cells to undergo apoptosis. A single local or i.p. injection of 213Bi-plasminogen activator inhibitor type 2 was able to completely regress the growth of tumours and lymph node metastases 2 days post subcutaneous inoculation, and obvious tumour regression was achieved in the therapy groups compared with control groups with 213Bi-plasminogen activator inhibitor type 2 when the tumours measured 30–40 mm3 and 85–100 mm3. All control animals and one of five (20%) mice treated with 3 mCi kg−1 213Bi-plasminogen activator inhibitor type 2 developed metastases in the lymph nodes while no lymphatic spread of cancer was found in the 6 mCi kg−1 treated groups at 2 days and 2 weeks post-cell inoculation. These results demonstrate that this novel 213Bi-plasminogen activator inhibitor type 2 conjugate selectively targets prostate cancer in vitro and in vivo, and could be considered for further development for the therapy of prostate cancer, especially for the control of micro-metastases or in minimal residual disease.British Journal of Cancer (2002) 86, 1197–1203. DOI: 10.1038/sj/bjc/6600179 www.bjcancer.com© 2002 Cancer Research UK

Three-dimensional type I collagen gel system for the study of osteoblastic metastases produced by metastatic prostate cancer.

We developed a three-dimensional type I collagen gel cell culture system that allows coculturing of human MG-63 osteoblast-like cells and various human cancer cells. Inoculation of human PC-3 metastatic prostate cancer cells into this type I collagen gel containing human MG-63 osteoblast-like cells produced an osteoblastic-like reaction that presented as an increased number of MG-63 cells and increased density of type I collagen around MG-63 cells adjacent to inoculated PC-3 cells by microscope analysis. Under identical experimental conditions, inoculation of cell-free medium, human KLE endometrial adenocarcinoma cells, and Calu-1 lung cancer cells did not produce this blastic-like reaction. In situ hybridization documented the uniform expression of insulin-like growth factor I (IGF-I) and of urokinase-type plasminogen activator (uPA) mRNA in MG-63 and PC-3 cells separately cultured in this substrata. The uniform expression of uPA was also documented by immunocytochemistry using a monoclonal and a polyclonal antihuman uPA antibody. The relative expression of uPA was higher in PC-3 cells than in MG-63, KLE, and Calu-1 cancer cells. We conclude that this novel cell culture system may become a useful model to study the pathophysiology of the osteoblastic reaction in vitro.