rottlerin and Prostatic-Neoplasms

Rottlerin as a natural agent, which is isolated from Mallotus philippinensis, has been identified to play a critical role in tumor inhibition. However, the molecular mechanism of rottlerin-mediated anti-tumor activity is still ambiguous. It has been reported that EZH2 exhibits oncogenic functions in a variety of human cancers. Therefore, inhibition of EZH2 could be a promising strategy for the treatment of human cancers. In this study, we aim to explore whether rottlerin could inhibit tumorigenesis via suppression of EZH2 in prostate cancer cells. Multiple approaches such as FACS, Transwell invasion assay, RT-PCR, Western blotting, and transfection were performed to determine our aim. We found that rottlerin treatment led to inhibition of cell growth, migration and invasion, but induction of apoptosis in prostate cancer cells. Importantly, we defined that rottlerin decreased the expression of EZH2 and H3K27me3 in prostate cancer cells. Moreover, overexpression of EZH2 abrogated the rottlerin-induced inhibition of cell growth, migration, and invasion in prostate cancer cells. Consistently, down-regulation of EZH2 enhanced rottlerin-triggered anti-tumor function. Collectively, our work demonstrated that rottlerin exerted its tumor suppressive function via inhibition of EZH2 expression in prostate cancer cells. Our findings indicated that rottlerin might be a potential therapeutic compound for treating patients with prostate cancer.

Topics: Acetophenones; Antineoplastic Agents, Phytogenic; Apoptosis; Benzopyrans; Cell Movement; Cell Proliferation; Down-Regulation; Enhancer of Zeste Homolog 2 Protein; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Invasiveness; PC-3 Cells; Prostatic Neoplasms; Signal Transduction

Chemotherapy is a vital therapeutic strategy for castration-resistant prostate cancer (CRPC). We have previously shown that NSC606985 (NSC), a camptothecin (CPT) analog, induced cell apoptosis via interacting with topoisomerase I (Topo I) in prostate cancer cells. In the present study, the effect and mechanism of CPT analogs in LAPC4 cells were investigated. LAPC-4 cells were treated with NSC, CPT, and topotecan. Cell proliferation, apoptosis, and protein kinase Cδ (PKCδ) subcellular activation were measured at different doses and time-points, with or without PKCδ inhibition or knockdown of PKCδ expression. NSC at doses ranging from 10 to 100 nM induced a dose-dependent increase in viable cell number and DNA biosynthesis with mild cell apoptosis, whereas, at doses ranging from 500 nM to 5 mM, NSC produced a dose-dependent decrease in cell proliferation and DNA biosynthesis with a significant induction of cell apoptosis. Both NSC-induced cell proliferation and apoptosis were blocked by knockdown of PKCδ with a specific RNAi, or by the co-administration of rottlerin, a PKCδ inhibitor. Moreover, NSC produced a dose-dependent subcellular activation of PKCδ. The dose-dependent dual action of NSC is mediated at least in part through the differential subcellular activation of PKCδ in LAPC4 cells. The demonstration of a differential cell response to camptothecin analogs would facilitate the identification of biomarker(s) to CPT sensitivity and promote the personalization of CPT chemotherapy in CRPC.

Topics: Acetophenones; Apoptosis; Benzopyrans; Camptothecin; Cell Line, Tumor; Cell Proliferation; DNA Topoisomerases, Type I; Dose-Response Relationship, Drug; Humans; Male; Precision Medicine; Prostatic Neoplasms; Protein Kinase C-delta

Autophagy plays an important role in cellular homeostasis through the disposal and recycling of cellular components. Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and drug resistance. Rottlerin (Rott) is an active molecule isolated from Mallotus philippinensis, a medicinal plant used in Ayurvedic Medicine for anti-allergic and anti-helminthic treatments, demonstrates anticancer activities. However, the molecular mechanisms by which it induces autophagy in prostate CSCs have not been examined. The main objective of the paper was to examine the molecular mechanisms by which Rott induces autophagy in prostate CSCs. Autophagy was measured by the lipid modification of light chain-3 (LC3) and the formation of autophagosomes. Apoptosis was measured by flow cytometer analysis. The Western blot analysis was used to examine the effects of Rott on the expression of PI3K, phosphorylation of Akt, phosphorylation of mTOR, and phosphorylation of AMPK in pros CSCs. RNAi technology was used to inhibit the expression of Beclin-1 and ATG-7. Rott induced the lipid modification of light chain-3 (LC3) and the formation of autophagosomes after 24h of Rott treatment in prostate CSCs. Rott-treated prostate CSCs induced transition from LC3-I to LC3-II, a hall mark of autophagy. Rott also induced the expression of Atg5, Atg7, Atg12 and Beclin-1 proteins during autophagy. The knock-down of Atg7 and Beclin-1 blocked Rott-induced autophagy. Furthermore, Rott induced AMPK phosphorylation was blocked by 3-MA, Baf and CHX. In addition, inhibition of AMPK expression by shRNA blocked Rott induced autophagy. In conclusion, a better understanding of the biology of autophagy and the pharmacology of autophagy modulators has the potential for facilitating the development of autophagy-based therapeutic interventions for prostate cancer.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Autophagy; Benzopyrans; Blotting, Western; Cell Line, Tumor; Flow Cytometry; Humans; Male; Phosphatidylinositol 3-Kinase; Prostatic Neoplasms; Signal Transduction; TOR Serine-Threonine Kinases

Activation of Wnt/β-catenin signaling can result in up-regulation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling. We found that rottlerin, a natural plant polyphenol, suppressed LRP6 expression and phosphorylation, and inhibited Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of rottlerin on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were confirmed in human prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells. Mechanistically, rottlerin promoted LRP6 degradation, but had no effects on LRP6 transcriptional activity. In addition, rottlerin-mediated LRP6 down-regulation was unrelated to activation of 5'-AMP-activated protein kinase (AMPK). Importantly, we also found that rottlerin inhibited mTORC1 signaling in prostate and breast cancer cells. Finally, we demonstrated that rottlerin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/β-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity with IC(50) values between 0.7 and 1.7 μM for prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells. Our data indicate that rottlerin is a novel LRP6 inhibitor and suppresses both Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells, and that LRP6 represents a potential therapeutic target for cancers.

Topics: Acetophenones; Antineoplastic Agents; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Screening Assays, Antitumor; Female; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Inhibitory Concentration 50; Low Density Lipoprotein Receptor-Related Protein-6; Male; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Promoter Regions, Genetic; Prostatic Neoplasms; Proteolysis; Survivin; TOR Serine-Threonine Kinases; Transcription, Genetic; Wnt Signaling Pathway; Wnt3A Protein

Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. The aim of this study was to investigate whether Bradykinin is associated with migration of prostate cancer cells.. Cancer cells migration activity was examined using the Transwell assay. The c-Src and PKCδ phosphorylation was examined by using Western blot method. The qPCR was used to examine the mRNA expression of metalloproteinase. A transient transfection protocol was used to examine NF-κB activity.. We found that bradykinin increased the chemomigration and the expression of MMP-9 of human prostate cancer cells. Bradykinin-mediated chemomigration and metalloproteinase expression was attenuated by PKCδ inhibitor (rottlerin), PKCδ siRNA, c-Src inhibitor (PP2) and c-Src mutant. Activations of PKCδ, c-Src and NF-κB pathways after bradykinin treatment was demonstrated, and bradykinin-induced expression of metalloproteinase and chemomigration activity was inhibited by the specific inhibitor and mutant of PKCδ, c-Src, and NF-κB cascades.. This study showed for the first time that the bradykinin mediates migration of human prostate cancer cells. One of the mechanisms underlying bradykinin directed migration was transcriptional up-regulation of MMP-9 and activation of B2 receptor, PKCδ, c-Src, and NF-κB pathways.

Topics: Acetophenones; Adenocarcinoma; Benzopyrans; Bradykinin; Cell Line, Tumor; Cell Movement; Humans; Male; Matrix Metalloproteinase 9; NF-kappa B; Phosphorylation; Prostatic Neoplasms; Protein Kinase C-delta; Receptor, Bradykinin B2; RNA, Small Interfering; Signal Transduction; src-Family Kinases

Combination therapy, which can optimize killing activity to cancers and minimize drug resistance, is a mainstream therapy against hormone-refractory prostate cancers (HRPCs). Rottlerin, a natural polyphenolic component, synergistically increased PC-3 (a HRPC cell line) apoptosis induced by camptothecin (a topoisomerase I inhibitor). Using siRNA technique to knockdown protein kinase C-δ (PKCδ), the data showed that rottlerin-mediated synergistic effect was PKCδ-independent, although rottlerin has been used as a PKCδ inhibitor. Rottlerin potentiated camptothecin-induced DNA fragmentation at S phase and ATM phosphorylation at Ser1981. The effect was correlated to apoptosis (r2 = 0.9). To detect upstream signals, the data showed that camptothecin acted on and stabilized topoisomerase I-DNA complex, leading to the formation of camptothecin-trapped cleavage complexes (TOP1cc). The effect was potentiated by rottlerin. To determine DNA repair capability, the time-related γH2A.X formation was examined after camptothecin removal. Consequently, rottlerin significantly inhibited camptothecin removal-mediated decline of γH2A.X formation at S phase, indicating the impairment of DNA repair activity in the presence of rottlerin. The combinatory treatment of camptothecin and rottlerin induced conformational change and activation of Bax and formation of truncated Bad, suggesting the contribution of mitochondria stress to apoptosis. In summary, the data suggest that rottlerin-mediated camptothecin sensitization is through the augmented stabilization of TOP1cc, leading to an increase of DNA damage stress and, possibly, an impairment of DNA repair capability. Subsequently, mitochondria-involved apoptosis is triggered through Bax activation and truncated Bad formation. The novel discovery may provide an anticancer approach of combinatory use between rottlerin and camptothecin for the treatment of HRPCs.

Topics: Acetophenones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzopyrans; Blotting, Western; Camptothecin; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; DNA Cleavage; DNA Topoisomerases, Type I; Drug Synergism; Flow Cytometry; Humans; In Situ Nick-End Labeling; Male; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Protein Kinase C-delta; RNA, Small Interfering; Topoisomerase I Inhibitors; Transfection

We studied the role of protein kinase C isoform PKCdelta in ceramide (Cer) formation, as well as in the mitochondrial apoptosis pathway induced by anticancer drugs in prostate cancer (PC) cells. Etoposide and paclitaxel induced Cer formation and apoptosis in PKCdelta-positive LNCaP and DU145 cells but not in PKCdelta-negative LN-TPA or PC-3 cells. In contrast, these drugs induced mitotic cell cycle arrest in all PC cell lines. Treatment with Rottlerin, a specific PKCdelta inhibitor, significantly inhibited drug-induced Cer formation and apoptosis in LNCaP cells, as did overexpression of dominant negative-type PKCdelta. Overexpression of wild-type PKCdelta had an opposite effect in PC-3 cells. Notably, etoposide induced biphasic Cer formation in LNCaP cells. The early and transient Cer increase resulted from de novo Cer synthesis, while the late and sustained Cer accumulation was derived from sphingomyelin hydrolysis by neutral sphingomyelinase (nSMase). Cer, in turn, induced mitochondrial translocation of PKCdelta and stimulated the activity of this kinase, promoting cytochrome c release and caspase-9 activation. Furthermore, the specific caspase-9 inhibitor LEHD-fmk significantly inhibited etoposide-induced nSMase activation, Cer accumulation, and PKCdelta mitochondrial translocation. These results indicate that PKCdelta plays a crucial role in activating anticancer drug-induced apoptosis signaling by amplifying the Cer-mediated mitochondrial amplification loop.

Topics: Acetophenones; Antineoplastic Agents, Phytogenic; Apoptosis; Benzopyrans; Caspase Inhibitors; Caspases; Cell Fractionation; Ceramides; Enzyme Inhibitors; Etoposide; Flow Cytometry; Humans; Isoenzymes; Male; Mitochondria; Paclitaxel; Prostatic Neoplasms; Protein Kinase C; Protein Kinase C-delta; Signal Transduction; Sphingomyelins; Tumor Cells, Cultured