Docetaxel In Prostate Cancer Cells Research Articles

Increasing the radiation-induced cytotoxicity by silver nanoparticles and docetaxel in prostate cancer cells.

Radiation therapy is utilized for treatment of localized prostate cancer. Nevertheless, cancerous cells frequently develop radiation resistance. While higher radiation doses have not always been effective, radiosensitizers have been extensively studied for their ability to enhance the cytotoxic effects of radiation.So, this study aims to evaluate the possible radiosensitization effects of docetaxel (DTX) and silver nanoparticles (SNP) in LNCaP cells. The cytotoxic effects of DTX, SNP and2Gy of X-Ray radiation treatments were assessed in human LNCaP cell line using the MTT test after 24 h. Moreover, the effects of DTX, SNP and radiation on Epidermal growth factor (EGF), Caspase 3, inducible nitric oxide synthase and E-cadherin gene expression were analyzed using the Real-time PCR method. The level of Hydrogen peroxide (H2O2), an oxidative stress marker, was also detected 24h after various single and combined treatments. The combinations of SNP (in low toxic concentration) and/or DTX (0.25× IC50 and 0.5 × IC50 concentrations for triple and double combinations respectively) with radiation induced significant cytotoxicity in LNCaP cells in comparison to monotherapies. These cytotoxic effects were associated with the downregulation of EGF mRNA. Additionally, H2O2 levels increased after Radiation + SNP + DTX triple combination and double combinations including Radiation + SNP and Radiation + DTX versus single treatments. The triple combination treatment also increased Caspase 3 and and E-cadherin mRNA levels in compared to single treatments in LNCaP cells. Our results indicate that the combination of SNP and DTX with radiation induces significant anti-cancer effects. Upregulation of Caspase 3 and E-cadherin gene expression, and decreased mRNA expression level of EGF may be exerted specifically by use of this combination versus single treatments.

Secreted miR-210-3p, miR-183-5p and miR-96-5p reduce sensitivity to docetaxel in prostate cancer cells

Docetaxel (DCT) resistance is one of the main factors responsible for treatment failure in metastatic prostate cancer (PCa). Although several mechanisms of DCT resistance have been elucidated, the issue is still far from comprehensive. In this work we show that miR-96-5p, miR-183-5p and miR-210-3p (referred to as sDCTR-miRNAs) are specifically released by DCT resistant (DCTR) PCa clones and decrease the efficacy of DCT in PCa cells when overexpressed. Through bioinformatic analysis, we identified several potential targets of sDCTR-miRNAs’ activity including FOXO1, IGFBP3, and PDCD4 known to exert a role in DCT resistance. Additionally, we found that PPP2CB and INSIG1 mediated the ability of sDCTR-miRNAs to reduce the efficacy of DCT. We explored whether secreted sDCTR-miRNAs could affect the phenotype of PCa cells. We found that exposure to exosomes derived from DCTR PCa clones (in which the content of sDCTR-miRNAs was higher than in exosomes from parental cells), as well as exposure to exosome loaded with sDCTR-miRNAs, reduced the cytotoxicity of DCT in PCa cells sensitive to the drug. Finally, we validated circulating miR-183-5p and miR-21-5p as potential predictive biomarkers of DCT resistance in PCa patients. Our study suggests a horizontal transfer mechanism mediated by exosomal miRNAs that contributes to reduce docetaxel sensitivity and highlights the relevance of cell-to-cell communication in drug resistance.

Polysaccharides from Lentinus edodes prevent acquired drug resistance to docetaxel in prostate cancer cells by decreasing the TGF-β1 secretion of cancer-associated fibroblasts.

Prostate cancer is one of the most prevalent lethal diseases among men globally. In the treatment of prostate cancer, the limited therapeutic efficacy of the standard non-hormonal systemic therapy docetaxel (DTX) represents an important challenge. Cancer-associated fibroblasts (CAFs) play a crucial role in resistance to therapy because of their prevalence and functional pleiotropy in tumor environments. Our previous research revealed that MPSSS, a novel polysaccharide extracted from Lentinus edodes, could significantly attenuate the immunosuppressive function of myeloid suppressor cells and CAFs. In this study, we investigated whether MPSSS could potentiate the efficacy of DTX against prostate cancer by inhibiting CAF-induced chemoresistance and elucidated its underlying mechanisms. The sensitivity of PC-3 prostate cancer cells cultured with conditioned medium derived from CAFs (CAF-CM) to DTX was assessed. The resistance effect induced by CAF-CM was abolished when CAFs were pretreated with MPSSS. Bioinformatic analysis of datasets from the Gene Expression Omnibus database revealed the activation of the transforming growth factor β1 (TGF-β1) signaling pathway in DTX-resistant cells. Based on this finding, we demonstrated that treatment with the TGF-β1 receptor inhibitor SB525334 reversed DTX resistance in CAFs, suggesting that TGF-β1 secreted by CAFs was a crucial intermediary in the development of DTX resistance in PC3 cells. Further research revealed that MPSSS decreases the secretion of TGF-β1 by inhibiting the JAK2/STAT3 pathway via Toll-like receptor 4 in CAFs. Overall, MPSSS might be a potential adjuvant treatment for DTX resistance in prostate cancer.

Combination Modality Using Quercetin to Enhance the Efficacy of Docetaxel in Prostate Cancer Cells.

The standard of care chemotherapy drug presently used to treat castration-resistant prostate cancer (CRPC), docetaxel (Doc), also develops chemoresistance, thereby reducing its clinical utility. Since resistance to chemotherapy drugs can be overcome by co-treatment with plant-based bio-active compounds we undertook the present study to evaluate if quercetin (Que), a flavonoid present in plants such as onions, apples, olives, and grapes can enhance the efficacy of Doc. We studied the separate and combined effects of Que and Doc at different doses and different combination approaches in two different prostate cancer cell lines, DU-145 (moderately aggressive) and PC-3 (very aggressive), and assessed the effects of these combinations on viability, proliferation, and apoptosis. Monotherapy with these drugs showed dose-dependent cytotoxicity; however, only Doc monotherapy showed a statistically significant difference in IC50 levels (IC50 = 4.05 ± 0.52 nM for PC-3 and IC50 = 2.26 ± 0.22 nM for DU-145). In combination treatment, we used three different treatment approaches (TAP). The concentrations and range analyzed were chosen based on the approximate cytotoxicity of 30-50% when the drugs were used individually. Our observations indicate that the most beneficial effect of the Que and Doc combination was obtained with the TAP-2 approach, which is pre-treatment with all doses of Que for 24 h followed by low doses of Doc for another 24 h. Using this approach, we observed synergism at low concentrations of Doc (0.5 and 1.0 nM) and all concentrations of Que. An additive effect was observed at moderate and high concentrations of Doc (1.5, 2.0, and 2.5 nM) and all concentrations of Que in both cell lines. The TAP-2 strategy was also helpful in overcoming Doc resistance in resistant CaP cells. In summary, Que improved the therapeutic effect of Doc in CRPC, and it is proposed that this improvement is mediated through multiple mechanisms. This study provides a novel therapeutic modality for an effective combination using Doc and Que to enhance the efficacy of Doc in an innocuous manner for Doc resistance and CRPC treatment.

A novel survivin dimerization inhibitor without a labile hydrazone linker induces spontaneous apoptosis and synergizes with docetaxel in prostate cancer cells

Survivin, a member of the inhibitor of apoptosis protein family, exists as a homodimer and is aberrantly upregulated in a wide spectrum of cancers. It was thought to be an ideal target due to its lack of expression in most adult normal tissues and importance in cancer cell survival. However, it has been challenging to target survivin due to its “undruggable” nature. We previously attempted to target its dimerization domain with a hypothesis that inhibiting survivin dimerization would promote its degradation in proteasome, which led to identification of a lead small-molecule inhibitor, LQZ-7F. LQZ-7F consists of a flat tetracyclic aromatic core with labile hydrazone linking a 1,2,5-oxadiazole moiety. In this study, we tested the hypothesis that LQZ-7F could be developed as a prodrug because the labile hydrazone linker could be hydrolyzed, releasing the tetracyclic aromatic core. To this end, we synthesized the tetracyclic aromatic core (LQZ-7F1) using reported procedure and tested LQZ-7F1 for its biological activities. Here we show that LQZ-7F1 has a significantly improved potency with submicromolar IC50’s and induces spontaneous apoptosis in prostate cancer cells. It also more effectively inhibits survivin dimerization and induces survivin degradation in a proteasome-dependent manner than LQZ-7F. We also show that the combination of LQZ-7F1 and docetaxel have strong synergism in inhibiting prostate cancer cell survival. Together, we conclude that the hydrazone linker with the oxadiazole tail is dispensable for survivin inhibition and the survivin dimerization inhibitor, LQZ-7F, may be developed as a prodrug for prostate cancer treatment and to overcome docetaxel resistance.

Solamargine Inhibits Prostate Cancer Cell Growth and Enhances the Therapeutic Efficacy of Docetaxel via Akt Signaling.

Prostate cancer (PCa) has become a leading cause of cancer-associated incidence and mortality in men worldwide. However, most primary PCas relapse to castration-resistant PCa (CRPC) after androgen deprivation treatment. The current treatment for CRPC is based on chemotherapeutic drugs such as docetaxel, while the development of chemoresistance and severe side effects limit the therapeutic benefit. Solamargine, a natural alkaloid isolated from a traditional Chinese herbal medicine known as Solanum nigrum, exhibits antitumor activity in various human cancers. In this study, we demonstrated that solamargine substantially inhibited CRPC cell growth in a dose-dependent manner through the suppression of phosphoinositide 3-kinase (PI3K)/Akt signaling. Moreover, solamargine exhibited significant antitumor effects in mouse xenograft models. Bioinformatics analysis of docetaxel-resistant PCa cells indicated that the PI3K/Akt pathway mediated the chemoresistance of CRPC. Furthermore, solamargine significantly enhanced the efficacy of docetaxel in PCa cells. These results reveal the therapeutic potential of solamargine against human PCa.

Silencing of circDPP4 suppresses cell progression of human prostate cancer and enhances docetaxel cytotoxicity through regulating the miR-564/ZIC2 axis.

Circular RNA derived from dipeptidyl peptidase 4 (circDPP4; ID: hsa_circ_0056881) is one of the top increased circRNAs in prostate cancer (PC) and docetaxel (DTX)-based chemotherapy is the primary therapeutic choice for PC. However, its repertoire in PC development and chemoresistance remains to be documented. Expression of circDPP4, microRNA (miR)-564, and zinc finger of the cerebellum 2 (ZIC2) was detected by a real-time quantitative polymerase chain reaction and western blotting; the direct interaction was validated by an RNA pull-down assay, a dual-luciferase reporter assay, and RNA immunoprecipitation. Cell progression was measured by a cell-counting kit-8 assay, colony formation assay, flow cytometry, a transwell assay, a xenograft experiment, and immunohistochemistry. DTX cytotoxicity was confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Expression of circDPP4 is upregulated in PC tumors from 60 patients and PC cell lines, and a higher circDPP4 might predict poor overall survival. Decreasing circDPP4 suppresses cell proliferation, colony formation, migration/invasion, and 50% inhibitory concentration of DTX in PC cells, and promotes the apoptosis rate. Both overexpressing miR-564 and inhibiting ZIC2 could imitate those effects, whereas inhibiting miR-564 and restoring ZIC2 could separately counteract them. Mechanistically, circDPP4 functions as a miR-564 sponge and regulates the expression of ZIC2, a target gene for miR-564. Tumor growth is retarded by silencing circDPP4, accompanied by elevated miR-564 and attenuated Ki-67 and ZIC2. Blocking circDPP4 antagonizes cell progression of PC and contributes to in vitro DTX cytotoxicity via regulating the miR-564/ZIC2 axis, at least. The present study suggests that circDPP4 is a potential biomarker and target for PC.

Oncolytic adenovirus carrying SPAG9-shRNA enhanced the efficacy of docetaxel for advanced prostate cancer.

Sperm-associated antigen 9 (SPAG9) is closely related to the growth and metastasis of advanced prostate cancer. Docetaxel (DTX) is the gold standard for chemotherapy of prostate cancer, but its side effects decrease the life quality of patients. Therefore, it is urgent to develop combination therapy to increase chemotherapy efficacy for advanced prostate cancer. Oncolytic adenovirus carrying a short hairpin RNA (shRNA) targeting SPAG9 (ZD55-shSPAG9) was applied alone or in combination with docetaxel in prostate cancer cells. Cells were analyzed by cell counting kit-8, Hocehst-33258, transwell and western blot analysis. For in vivo experiments, nude mice were loaded with prostate cancer cells. ZD55-shSPAG9 effectively silenced the expression of SPAG9 in prostate cancer cells in vitro and in vivo. The replication of ZD55-shSPAG9 in prostate cancer cells was not affected by docetaxel, but the combined use of ZD55-shSAPAG9 and docetaxel has a better inhibitory effect on tumor growth and invasion in vitro and in vivo. Our study showed that the combined use of ZD55-shSPAG9 and docetaxel may be a new approach to the treatment of advanced prostate cancer.

The lncRNA OGFRP1/miR-149-5p/IL-6 axis regulates prostate cancer chemoresistance

BackgroundThe long non-coding RNA (lncRNA) OGFRP1 has been found to promote malignancy in prostate cancer (PC) and other cancer types. How this lncRNA functions in the regulation of PC chemoresistance, however, is poorly defined. MethodsqRT-PCR was employed to measure OGFRP1, miR-149-5p, and IL-6 expression in PC tissues and cells. IC50 values for paclitaxel and docetaxel in PC cells were assessed via a CCK-8 assay approach. Putative miR-149-5p binding targets were identified and validated through bioinformatics assays and luciferase reporter assays, respectively. The impact of OGFRP1 on PC chemoresistance in vivo was validated using a xenograft model system. ResultsDocetaxel-resistant PC (PC/DR) cells and tissues exhibited reduced OGFRP1 expression and increased miR-149-5p expression. Knocking down OGFRP1 augmented the sensitivity of these PC cells to docetaxel and paclitaxel in vitro and in vivo. Mechanistically, OGFRP1 was found to bind and sequester miR-149-5p within PC/DR cells, thereby indirectly regulating IL-6 expression. Consistent with this model, the overexpression of IL-6 reversed the OGFRP1 knockdown-mediated reductions in docetaxel and paclitaxel IC50 values for these PC cells. ConclusionsOGFRP1 can sequester miR-149-5p, thereby indirectly promoting IL-6 upregulation and thereby promoting chemoresistance in PC cells. This OGFRP1/miR-149-5p/IL-6 axis may thus be a promising target for therapeutic efforts aimed at PC chemosensitization and treatment.

Protein Disulfide Isomerase 4 Drives Docetaxel Resistance in Prostate Cancer

Background: Protein disulfide isomerase 4 (PDIA4) has been reported to be closely associated with chemoresistance in several types of malignancies. But the pathogenic mechanisms of PDIA4 involved in docetaxel (DTX) resistance in prostate cancer (PCa) are still unknown. Hence, this study was conducted to evaluate the potential effect of PDIA4 on chemoresistance to DTX in PCa cells and to investigate the underlying mechanisms. Methods: Two types of DTX-resistant PCa cells, that is, DTX-resistant PC-3 cells (PC-3/DTXR) and C4-2B cells (C4-2B/DTXR) were developed, as well as the parental PC-3 and C4-2B cells were obtained to investigate these issues. Short hairpin RNAs targeting human PDIA4 to knockdown the expression of PDIA4 or PDIA4-expressing adenoviral vectors to overexpress the PDIA4 were transfected into PCa cells to study the underlying mechanisms of PDIA4 involving in PCa DTX resistance. Results: Results showed that PDIA4 exhibited a dramatic overexpression in PC-3/DTXR and C4-2B/DTXR cells. Down-regulation of PDIA4 by infecting PC-3/DTXR and C4-2B/DTXR cells with shPDIA4 lentivirus stimulated cell death by prompting apoptosis. Up-regulation of PDIA4 by infecting PC-3 and C4-2B cells with PDIA4-expressing adenovirus showed severer resistance to DTX. In addition, PDIA4 up-regulation induced phosphorylated protein kinase B (Akt) expression, while PDIA4 knockdown significantly inhibited the expression in PCa cells. Conclusions: Our study indicates that PDIA4 is a negative regulator of PCa cell apoptosis and plays a critical role in PCa DTX resistance by activating the Akt-signaling pathway. Thereby, it implies that targeting PDIA4 could be a potential adjuvant therapeutic approach against DTX resistance in PCa.

Upregulation of GRIM‐19 augments the sensitivity of prostate cancer cells to docetaxel by targeting Rad23b

The gene associated with retinoid-interferon mortality (GRIM-19) has been reported to be correlated with drug resistance, whereas its functional role in prostate cancer (PC) is not fully understood. This study aims to clarify the potential role and molecular mechanisms of GRIM-19 on the response of PC cells to chemical drug docetaxel. mRNA and protein level of GRIM-19 expression in cells and tissues of PC were measured by quantitative real-time PCR and western blot, respectively. Knock-down of GRIM-19 in PC cells was performed using siRNA. Cell apoptosis was determined by flow cytometric analysis. DNA damage in PC cells was detected by γ-H2AX staining. GRIM-19 was downregulated in PC tissues and cell lines. Knock-down of GRIM-19 increased the resistance of PC cells to docetaxel, and overexpression of GRIM-19 promoted docetaxel-induced apoptotic death in PC cells. Mechanistically, GRIM-19 downregulated the expression of the survival gene Rad23b, which promoted DNA damage repair. Overexpression of Rad23b reversed GRIM-19-mediated response to docetaxel in PC cells. GRIM-19 promoted the sensitivity of PC cells to docetaxel by downregulating Rad23b, which may serve as a promising target to develop a better strategy of chemotherapy for PC.

Blockade of γ-Glutamylcyclotransferase Enhances Docetaxel Growth Inhibition of Prostate Cancer Cells.

γ-Glutamylcyclotransferase (GGCT) is highly expressed in many forms of cancer, and is a promising therapeutic target. The present study investigated whether inhibition of GGCT enhanced the antiproliferative effects of the drug docetaxel in prostate cancer cells. Immunohistochemistry and western blot analysis were conducted to measure GGCT expression in prostate cancer tissue samples and cell lines. GGCT was inhibited using RNAi and a novel enzymatic inhibitor, pro-GA, and cell proliferation was evaluated with single and combination treatments of GGCT inhibitors and docetaxel. GGCT was highly expressed in cultured prostate cancer cells and patient samples. GGCT inhibition alone inhibited prostate cancer cell line proliferation and induced cellular senescence. GGCT inhibition in combination with apoptosis-inducing docetaxel had more potent antiproliferative effects than either drug used alone. GGCT inhibition may potentiate anticancer drug efficacy.

AICAR Induces Apoptosis and Inhibits Migration and Invasion in Prostate Cancer Cells Through an AMPK/mTOR-Dependent Pathway.

Current clinical challenges of prostate cancer management are to restrict tumor growth and prohibit metastasis. AICAR (5-aminoimidazole-4-carbox-amide-1-β-d-ribofuranoside), an AMP-activated protein kinase (AMPK) agonist, has demonstrated antitumor activities for several types of cancers. However, the activity of AICAR on the cell growth and metastasis of prostate cancer has not been extensively studied. Herein we examine the effects of AICAR on the cell growth and metastasis of prostate cancer cells. Cell growth was performed by MTT assay and soft agar assay; cell apoptosis was examined by Annexin V/propidium iodide (PI) staining and poly ADP ribose polymerase (PARP) cleavage western blot, while cell migration and invasion were evaluated by wound-healing assay and transwell assay respectively. Epithelial–mesenchymal transition (EMT)-related protein expression and AMPK/mTOR-dependent signaling axis were analyzed by western blot. In addition, we also tested the effect of AICAR on the chemosensitivity to docetaxel using MTT assay. Our results indicated that AICAR inhibits cell growth in prostate cancer cells, but not in non-cancerous prostate cells. In addition, our results demonstrated that AICAR induces apoptosis, attenuates transforming growth factor (TGF)-β-induced cell migration, invasion and EMT-related protein expression, and enhances the chemosensitivity to docetaxel in prostate cancer cells through regulating the AMPK/mTOR-dependent pathway. These findings support AICAR as a potential therapeutic agent for the treatment of prostate cancer.

PSMA-Targeted Mesoporous Silica Nanoparticles for Selective Intracellular Delivery of Docetaxel in Prostate Cancer Cells

Although docetaxel is currently broadly used in prostate cancer treatment, poor water solubility and systemic toxicity limit the dose and duration of therapy. In this context, although different na...

Abstract 4400: Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant prostate cancer cells

Abstract Introduction and objectives: It is well known as Warburg effect that anaerobic glycolytic pathway is activated in various type of advanced cancers including prostate cancer (PC). Lactate dehydrogenase-A (LDH-A) controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. Since LDH-A pathways have been implicated in chemoresistance in various cancers, we investigated whether inhibition of LDH-A pathway could mediate the sensitivity to docetaxel (DOC) in human PC cells. Materials and Method: Four PC cell lines (PC3, DU145, LNCaP, LN-CSS ) were used. LN-CSS is one of the LNCaP-derived castration-resistant PC (CRPC) cell lines established in our laboratories. Sodium oxamate (SO) was used as a specific LDH-A inhibitor. The protein expression was detected by western blot analysis using specific antibodies. Cell growth and survival were evaluated by WST-1 assays. Cell cycle progression and apoptotic inducibility were evaluated by flow cytometry using propidium iodide and Annexin V. The cytotoxicity of SO/DOC combination on PC cells was evaluated using the Chou-Talalay combination index (CI) method which offers quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations. Result: Western blot analysis showed that LDH-A protein was highly expressed in LN-CSS cells compared with other PC cell lines including the parental LNCaP. WST-1 assays showed that treatment with SO (50 mM) for 72 hours in PC cells resulted in growth inhibition (PC3; ~30%, DU145; ~55%, LNCaP; ~20%,LN-CSS; ~55% ), while SO has little growth inhibitory effects on normal lymphocytes in the concentrations between 1-100 mM. IC50 to DOC in PC cells were showed to be 4 nM, 1 nM, 1nM, and 4.5 nM in PC3, DU145, LNCaP and LN-CSS, respectively, suggesting that both PC3 and LN-CSS were relatively resistant to DOC compared with DU145 and LNCaP. Synergistic cytotoxicity was observed after the combination therapy with DOC and SO in LN-CSS (CI: 0.5) but not in PC3 (CI: 1.9), DU145 (CI: 2.0), or LNCaP (CI: 6.5). Cell cycle analyses revealed that the combination with DOC and SO for 72 hours resulted in the accumulation of cells in G2-M phase followed by sub-G1 accumulation in LN-CSS cells. Annexin V assays showed that 43% apoptosis was induced by the combination therapy in LN-CSS cells, while only 12% by DOC only in LN-CSS cells. Conclusion: Our results strongly suggest that LDH-A plays an important role in DOC resistance in advanced PC cells and inhibition of LDH-A promotes DOC-sensitivity especially in CRPC cells. Our study may provide valuable information for the future development of targeted therapies in patients with CRPC. Citation Format: Hiroyuki Muramatsu, Makoto Sumitomo, Shingo Morinaga, Hiroshi Saiki, Ikuo Kobayashi, Keishi Kajikawa, Genya Nishikawa, Yoshiharu Kato, Masahito Watanabe, Kent Kanao, Kogenta Nakamura, kazuhiro Yoshikawa. Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant 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 4400. doi:10.1158/1538-7445.AM2017-4400

Resveratrol induces cell cycle arrest and apoptosis with docetaxel in prostate cancer cells via a p53/ p21WAF1/CIP1 and p27KIP1 pathway.

Resveratrol (RES) is the most effective natural products used for the treatment of a variety of cancers. In this study, we tested the effect of RES in enhancing the efficacy of docetaxel (DTX) treatment in prostate cancer (PCa) cells. The C4-2B and DU-145 cell lines were treated with RES, DTX and combination followed by evaluating the apoptosis and cell cycle progression. The combined drug treatment up-regulates the pro-apoptotic genes (BAX, BID, and BAK), cleaved PARP and down regulates the anti-apoptotic genes (MCL-1, BCL-2, BCL-XL) promoting apoptosis. In C4-2B cells the combination up regulated the expression of p53, and cell cycle inhibitors (p21WAF1/CIP1, p27KIP), which, in turn, inhibited the expression of CDK4, cyclin D1, cyclin E1 and induced hypo-phosphorylation of Rb thus blocking the transition of cells in the G0/G1 to S phase. In contrast, the synergistic effect was not profound in DU145 due to its lesser sensitivity to DTX. The suppression of cyclin B1 and CDK1 expression in both cell lines inhibits the further progression of cells in G2/M phase. The current study demonstrates that combination treatment blocks the cell cycle arrest by modulation of key regulators and promotes apoptosis via p53 dependent and independent mechanism in PCa.

Molecular complexity of taxane-induced cytotoxicity in prostate cancer cells

BackgroundTaxanes are routinely used to treat men with advanced prostate cancer, yet their molecular mode of action is poorly characterized. Taxanes stabilize microtubules and may hence interfere with a plethora of cellular processes, most notably mitosis. However, prostate cancer is typically a slowly growing tumor suggesting that additional processes play a role in the response to taxanes. MethodsHere, we analyzed the potential effect of taxanes on microtubuli-dependent intracellular transport and signaling processes, specifically, nuclear translocation of the androgen receptor and modulation of the RAS-RAF-MEK-ERK signaling cascade. ResultsWe show that the androgen-driven nuclear translocation of the androgen receptor remains virtually undisturbed by docetaxel in prostate cancer cells. However, we found a striking down-regulation of activated ERK1/2 together with enhanced cytotoxicity in both docetaxel or cabazitaxel-treated cells that was comparable to direct MEK kinase inhibition. Remarkably, MEK inhibition alone was less effective in inducing cytotoxicity than taxanes indicating that a down-regulation of activated ERK1/2 may be necessary but is not sufficient for taxane-induced antitumoral effects. In line with this notion, we show in a xenograft mouse model that prostate cancer cells that are resistant to docetaxel overexpress activated ERK1/2. Taken together, our findings underscore that the modulation of ERK1/2 activation, in concert with other mechanisms, plays an important role in taxane-induced antineoplastic effects on prostate cancer cells. ConclusionsThese results suggest at least partially nonoverlapping effects of docetaxel and androgen deprivation therapy and hence help to understand recent clinical findings. A further elucidation of the mode of action of docetaxel would have important implications to optimize current treatment strategies and biomarker development for men with metastatic prostate cancer.

Abstract 405: Kindlin-2 regulates integrin function and sensitivity to docetaxel in prostate cancer cells

Abstract Over the past decade, many lines of evidence have emerged to implicate the three members of the kindlin family of FERM domain proteins as important regulators of integrin adhesion receptors. Specifically, kindlins appear to cooperate with talin to be essential regulators of the ligand binding function of integrins and thereby enhance the adhesive and migratory responses of cells. Kindlin-2 (FERMT2) is the most broadly distributed of the kindlins. To explore the relationship between integrin activation and kindlin-2, prostate cancer cell lines were screened and LNCaP prostate cancer cells were found to express relatively low levels of kindlin-2 and endogenous levels of beta-1 integrin on their surface. A portion of this integrin was expressed in an activated state as detected with an activation specific antibody, HUTS-4. These cells were transfected with vectors for kindlin-2, with or without talin-head domain, a combination that leads to activation of β3 integrins in model cells. Transfections with kindlin-2 suppressed β1 integrin activation on the surface of the LNCaP cells. This inhibitory effect was abolished by mutation of kindlin-2 that prevented its binding to integrin cytoplasmic tail. This inhibitory response to kindlin-2 on β1 integrin was in stark contract to the effects of kindlin-2 plus talin-head expression on β3 function, where co-expression of the same constructs enhanced integrin activation. A mutation in kindlin-2, deletion of its last 15 amino acids, that blocks activation of β3 integrins without preventing its binding to the integrin, also prevents its inhibition of β1 function. We next looked at the effects of kindlin-2 on docetaxel induced apoptosis as monitored with Annexin V staining and cell death as monitored by propidium iodide staining (both by flow cytometry). Compared to LNCaP cells expressing EGFP alone, cells overexpressing EGFP-kindlin-2 were protected against apoptosis and cell death. This effect was dependent upon binding of kindlin-2 to the cytoplasmic tail of integrins. Hence, the capacity of kindlin-2 to influence integrin function depends upon the integrin subfamily and can range from activation to suppression of function. Kindlin-2, which is often overexpressed in transformed cells, may desensitize cells towards chemotherapeutic agents. Citation Format: Edward F. Plow, Mitali Das, Jamila Hirbawi, Khalid Sossey-Alaoui. Kindlin-2 regulates integrin function and sensitivity to docetaxel in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 405. doi:10.1158/1538-7445.AM2015-405

GLIPR1-ΔTM synergizes with docetaxel in cell death and suppresses resistance to docetaxel in prostate cancer cells.

BackgroundDocetaxel is the first chemotherapy agent approved for treatment of metastatic castration-resistant prostate cancer (mCRPC). The limited survival benefit associated with the quick emergence of resistance and systemic toxicity diminished its efficacy. JNK-mediated apoptosis is one of the mechanisms of docetaxel activity whereas ERK1/2-c-Myc-CXCR4 signaling is implicated in the development of resistance and induction of migration. The aim of this study was to evaluate the hypothesis that the combination treatment with docetaxel and GLIPR1-ΔTM will synergistically induce greater cell death and inhibit the emergence of resistance and development of metastatic potential in prostate cancer (PCa) cells.MethodsThe synergistic effects of the docetaxel and GLIPR1-ΔTM were evaluated with DNA fragmentation, DAPI staining and MTS using paired t-test and isobologram study. The effects of the drugs on JNK and ERK1/2-c-Myc-CXCR4 signaling were evaluated with Western blot, DNA fragmentation, and MTS assays using the JNK inhibitor SP600125, and CXCR4 siRNA. The results of docetaxel and GLIPR1-ΔTM combination on migration were examined with scratch assay using the CXCR4 inhibitor AMD3100 while our hypothesis was examined in vivo using VCaP orthotopic xenograft model.ResultsWe found that GLIPR1-ΔΤΜ synergized with docetaxel to induce apoptosis in VCaP and PC-3 PCa cells through induction of JNK signaling and concomitant inhibition of ERK1/2-c-Myc-CXCR4 signaling. We showed that JNK activation mediates the apoptotic effects of the drug combination and that CXCR4 knockdown increases its efficacy. We also found that the addition of GLIPR1-ΔΤΜ to docetaxel decreases the migration of VCaP and PC-3 cells. The combination treatment with docetaxel and GLIPR1-ΔTM inhibited tumor growth and decreased metastatic potential in VCaP xenografts more than single agents did.ConclusionsOur data suggested that addition of GLIPR1-ΔTM treatment in PCa cells increases the efficacy of docetaxel and may inhibit the emergence of drug resistance; potentially permitting a decrease of docetaxel dose for patients with mCRPC eliminating its systemic toxicities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0395-0) contains supplementary material, which is available to authorized users.

Sensitization to docetaxel in prostate cancer cells by green tea and quercetin

Chemotherapy with docetaxel (Doc) is a standard treatment for metastatic and castration-resistant prostate cancer. However, chemoresistance and side effects of Doc limit its clinical success. We investigated whether natural products green tea (GT) and quercetin (Q), a flavonoid from apples and onions, will enhance the efficacy of Doc in androgen-independent (AI) prostate cancer cells. Two cell lines including LAPC-4-AI and PC-3 were treated in vitro with 40 μM of (−)-epigallocatechin gallate (EGCG), 5 μM of Q, 2 or 5 nM of Doc alone or in combination. The mixture of EGCG+Q+Doc increased the antiproliferative effect by threefold in LAPC-4-AI cells and eightfold in PC-3 cells compared to Doc alone. EGCG, Q and Doc in combination significantly enhanced cell cycle arrest at G2/M phase and increased apoptosis in both LAPC-4-AI and PC-3 cells compared to Doc alone. The mixture increased the inhibition of PI3K/Akt and the signal transducer and activator of transcription (Stat) 3 signaling pathways compared to Doc alone, and decreased the protein expression of multidrug resistance-related protein. In addition, the combination with EGCG and Q increased the inhibition of tumor cell invasion and colony formation in both LAPC-4-AI and PC-3 cells compared to Doc alone, and decreased the percentage of CD44+/CD24− stem-like LAPC-4-AI cells. In summary, GT and Q enhanced the therapeutic effect of Doc in castration-resistant prostate cancer cells through multiple mechanisms including the down-regulation of chemoresistance-related proteins. This study provides a novel therapeutic modality to enhance the efficacy of Doc in a nontoxic manner.