Andrographolide targets androgen receptor pathway in castration-resistant prostate cancer.

Androgen receptor (AR) signaling not only plays a pivotal role in the development of androgen-dependent prostate cancer but is also important in the growth and survival of castration-resistant prostate cancer (CRPC). The first line of treatment of androgen-dependent prostate cancer is the use of androgen deprivation therapy. However, most patients will eventually relapse due to development of CRPC. Thus, development of a strategy to target AR for treatment of CRPC is urgently needed. The authors have previously identified andrographolide as an inhibitor of interleukin-6, which can suppress tumor growth of prostate cancer cells by screening compounds from the Prestwick Natural compound library. In this study, they identified that andrographolide can inhibit AR expression and prostate cancer cell growth and induce apoptosis. Andrographolide is able to down-regulate AR expression at both mRNA and protein levels, prevents its nuclear translocation, and inhibits transactivation of its target genes. Andrographolide prevents the binding of Hsp90 to AR, resulting in proteasome-mediated AR degradation. Furthermore, andrographolide inhibits castration-resistant C4-2 cell growth by reducing AR expression and activity. Thus, andrographolide can be developed as a potential therapeutic agent for prostate cancer by inhibition of androgen receptor signaling.

Transcriptional regulation of histidine biosynthesis genes in Corynebacterium glutamicum.

Corynebacterium glutamicum, a gram-positive bacterium, has been widely used for industrial amino acid production. Corynebacterium glutamicum his genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. The latter his operon starts the transcription at the C residue localized 196 bp upstream of the hisD ATG start codon. Our computer-based sequence analysis showed that the region corresponding to the untranslated 5' end of the transcript, named the hisD leader region, displays the typical features of the T-box transcriptional attenuation mechanism. Therefore, expression of the cat reporter gene under the control of the wild-type or mutated hisD leader regions was tested in multi-copy (pProm and pTer series) and in single-copy (pInt series) systems under conditions of sufficient or limited histidine. Our mutational studies led to the conclusion that the CAU histidine specifier and 5'-UGGA-3' sequence in the hisD leader region are required for the hisDCB-orf1-orf2-hisHA-impA-hisFI gene regulation. The cat gene expression from the wild-type leader region was negatively regulated by histidine. However, the cat gene expression from mutated leader regions was irresponsive to the level of histidine in the growth medium. Taken together, we propose that a T-box mediated attenuation mechanism is responsible for the gene expression of the hisDCB-orf1-orf2-hisHA-impA-hisFI operon in C. glutamicum.

Sanguinarine suppresses prostate tumor growth and inhibits survivin expression.

Prostate cancer is a frequently occurring disease and is the second leading cause of cancer-related deaths of men in the United States. Current treatments have proved inadequate in curing or controlling prostate cancer, and a search for agents for the management of this disease is urgently needed. Survivin plays an important role in both progression of castration-resistant prostate cancer and resistance to chemotherapy. Altered expression of survivin in prostate cancer cells is associated with cancer progression, drug/radiation resistance, poor prognosis, and short patient survival. In the present study, the authors performed a cell-based rapid screen of the Prestwick Chemical Library consisting of 1120 Food and Drug Administration-approved compounds with known safety and bioavailability in humans to identify potential inhibitors of survivin and anticancer agents for prostate cancer. Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified as a novel inhibitor of survivin that selectively kills prostate cancer cells over "normal" prostate epithelial cells. The authors found that sanguinarine inhibits survivin protein expression through protein degradation via the ubiquitin-proteasome system. Sanguinarine induces apoptosis and inhibits growth of human prostate cancer cells and in vivo tumor formation. Administration of sanguinarine, beginning 3 days after ectopic implantation of DU145 human prostate cancer cells, reduces both tumor weight and volume. In addition, sanguinarine sensitized paclitaxel-mediated growth inhibition and apoptosis, offering a potential therapeutic strategy for overcoming taxol resistance. These results suggest that sanguinarine may be developed as an agent either alone or in combination with taxol for treatment of prostate cancer overexpressing survivin.

Andrographolide, an herbal medicine, inhibits interleukin-6 expression and suppresses prostate cancer cell growth.

Elevated interleukin-6 (IL-6), a major mediator of the inflammatory response, has been implicated in androgen receptor (AR) activation, cellular growth and differentiation, plays important roles in the development and progression of prostate cancer, and is a potential target in cancer therapy. Through drug screening using human prostate cancer cells expressing IL-6 autocrine loop, we found that andrographolide, a diterpenoid lactone isolated from a traditional Chinese and Indian medicinal plant Andrographis paniculata, could inhibit IL-6 expression and suppress IL-6-mediated signals. Andrographolide inhibits IL-6 expression at both mRNA and protein levels in a dose-dependent manner. Andrographolide suppresses both IL-6 autocrine loop- and paracrine loop-induced cell signaling including Stat3 and Erk phosphorylation. Furthermore, andrographolide inhibits cell viability and induces apoptotic cell death in both androgen-stimulated and castration-resistant human prostate cancer cells without causing significant toxicity to normal immortalized prostate epithelial cells. Moreover, treatment of andrographolide to mice bearing castration-resistant DU145 human prostate tumors that express constitutive IL-6 autocrine loop significantly suppresses tumor growth. Taken together, these results demonstrate that andrographolide could be developed as a therapeutic agent to treat both androgen-stimulated and castration-resistant prostate cancer possibly by suppressing IL-6 expression and IL-6-induced signaling.

Interleukin-6 regulates androgen synthesis in prostate cancer cells.

PURPOSE: The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor and stimulate tumor growth, suggesting that prostate cancer cells may survive androgen deprivation therapies by increasing intracrine androgen synthesis within the prostate. However, factors that regulate de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of androgen receptor activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 regulates intraprostatic androgen synthesis in prostate cancer cells. EXPERIMENTAL DESIGN: Quantitative reverse transcription-PCR and Western blotting were done to detect expression levels of steroidogenic enzymes. AKR1C3 promoter reporter was constructed and analyzed for IL-6-mediated AKR1C3 transcriptional activity. IL-6-mediated signaling was knocked down using small interfering RNA specific to IL-6 receptor and gp130, and the effect on AKR1C3 expression was examined. Intraprostatic androgen levels in prostate cancer cells in culture and in tumors were measured by an enzyme immunoassay (Testosterone EIA kit). RESULTS: We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes, including HSD3B2 and AKR1C3, involved in androgen biosynthesis. Down-regulation of IL-6 receptor and gp130 expression using specific small interfering RNA abolished IL-6-mediated AKR1C3 expression, suggesting that IL-6 signaling is responsible for AKR1C3 expression. IL-6 increases AKR1C3 promoter activity, indicating that the increase in IL-6-mediated AKR1C3 expression is in part at the transcriptional level. Treatment of IL-6 increased testosterone level in LNCaP cells. The tumor testosterone levels were detected at 378 pg/g in tumors generated from IL-6-overexpressing LNCaP-IL6(+) cells inoculated orthotopically into the prostates of castrated male nude mice. CONCLUSIONS: These results suggest that IL-6 increases levels of intracrine androgens through enhanced expression of genes mediating androgen metabolism in prostate cancer cells.

Interleukin-6 increases prostate cancer cells resistance to bicalutamide via TIF2.

The standard treatment for advanced, androgen-responsive prostate cancer is androgen deprivation therapy with or without a nonsteroidal antiandrogen, such as bicalutamide. Although maximal androgen blockade exhibits favorable responses in the majority of patients, prostate cancer eventually progresses to an androgen-refractory stage. The mechanism underlying bicalutamide resistance in the course of prostate cancer progression is incompletely understood. However, interleukin-6 (IL-6) plays a critical role in the development and progression of CRPC. Herein, we explored an association between IL-6 and bicalutamide resistance. To study this, series of lower and higher passages of LNCaP cell sublines generated by long-term exposure to IL-6 were used. The cells from higher passages of LNCaP treated with IL-6 developed resistance to bicalutamide treatment compared with parental LNCaP cells. The levels of transcriptional intermediary factor 2 (TIF2) in IL-6-treated LNCaP cells were found to be significantly higher than parental LNCaP cells. Down-regulation of TIF2 expression via short hairpin RNA in IL-6-treated LNCaP cells sensitized these cells to bicalutamide treatment, whereas overexpression of TIF2 in the parental LNCaP cells increased resistance to bicalutamide. Furthermore, overexpression of IL-6 attenuated bicalutamide-mediated blockage of androgen-induced androgen receptor nuclear translocation and recruitment. These results show that overexpression of IL-6 increases the resistance of prostate cancer cells to bicalutamide via TIF2. Overexpression of IL-6 not only plays an important role in prostate cancer progression but also contributes to bicalutamide resistance. Our studies suggest that bicalutamide-IL-6-targeted adjunctive therapy may lead to a more effective intervention than bicalutamide alone.

Interleukin-4 activates androgen receptor through CBP/p300.

BACKGROUND: Aberrant activation of androgen receptor (AR) plays an important role in the progression of castration resistant prostate cancer. Interleukin-4 (IL-4) enhances AR activation in the absence of androgen and stimulates castration resistant growth of androgen-sensitive prostate cancer cells. However, the mechanism of IL-4 mediated AR activation has not yet been revealed. METHODS: The effect of IL-4 on CBP/p300 expression was examined by Western blot analysis. The effect of IL-4 on the interactions of AR and CBP/p300 was examined by co-immunoprecipitation and ChIP assays. CBP/p300 siRNA was used to knockdown CBP/p300 expression to examine the role of CBP/p300 expression on IL-4 mediated AR activation. RESULTS: We found that IL-4 increases CBP/p300 protein expression and enhances interaction of AR with CBP/p300 proteins through an increase in the recruitment of CBP/p300 protein to the androgen responsive elements in the promoters of androgen responsive genes. Down regulation of CBP/p300 expression using CBP/p300 specific siRNA abolished IL-4 mediated AR activation, suggesting that CBP/p300 is responsible for AR activation induced by IL-4. Furthermore, AR activation can be enhanced by AR acetylation induced by IL-4 in prostate cancer cells. The IL-4 mediated AR acetylation can be blocked by knocking down CBP/p300 expression using CBP/p300 specific siRNA. CONCLUSION: These results suggest that IL-4 activates AR through enhanced expression of CBP/p300 and its histone acetyltransferase activity.

NF-kappaB2/p52 enhances androgen-independent growth of human LNCaP cells via protection from apoptotic cell death and cell cycle arrest induced by androgen-deprivation.

PURPOSE: Androgen-deprivation therapy only causes a temporary regression of prostate cancer, as all tumors will eventually progress to refractory to hormonal therapy after 1-3 years of treatment. The underlying mechanisms of prostate cancer androgen refractory progression are incompletely understood. In this study, we employed in vitro as well as in vivo models to examine the role of NF-kappaB2/p52 in prostate cancer growth and androgen independent progression. EXPERIMENTAL DESIGN: The effects of NF-kappaB2/p52 on cell growth, androgen responsiveness, cell cycle and apoptosis were examined in androgen sensitive LNCaP cells. The effect of NF-kappaB2/p52 on tumor growth was examined in intact and castrated male mice. RESULTS: Overexpression of NF-kappaB2/p52 enhances androgen-sensitive LNCaP human prostate cancer cell growth and clonogenic ability in androgen-deprived condition in vitro. NF-kappaB2/p52 induced androgen-independent growth is through protecting LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. In addition, NF-kappaB2/p52 stimulates Cyclin D1 expression and knock down of Cyclin D1 expression by siRNA abolished NF-kappaB2/p52-induced cell growth in vitro. Adenoviral mediated NF-kappaB2/p52 expression in LNCaP cells enhances tumor growth in intact male nude mice and induces tumor growth in castrated male nude mice, suggesting that overexpression of NF-kappaB2/p52 induces androgen-independent growth of androgen-sensitive LNCaP cells. CONCLUSIONS: Overexpression of NF-kappaB2/p52 protects androgen sensitive LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. NF-kappaB2/p52 activation induces androgen-independent growth in vitro and in vivo.

Interleukin-4 stimulates androgen-independent growth in LNCaP human prostate cancer cells.

BACKGROUND: Clinical data showed that the levels of interleukin-4 (IL-4) are significantly elevated in serum of patients with ablation resistant prostate cancer. Previous studies demonstrated that IL-4 enhances androgen receptor (AR) activation mediated by NF-kappaB in the absence or in the very low levels of androgen in prostate cancer cells. In this study, the role of IL-4 in promoting the growth of androgen-independent prostate cancer cells was examined. METHODS: LNCaP cells were transfected with a full-length IL-4 cDNA and stable clones expressing IL-4 were selected. The growth of LNCaP cells expressing IL-4 was analyzed in vitro and in vivo both in the presence and absence of androgen. RESULTS: Overexpression of IL-4 enhances the growth of androgen-sensitive LNCaP cells in culture media containing charcoal-stripped FBS condition (CS-FBC), and increases the sensitivity of LNCaP cells in response to androgen stimulation. The DHT-mediated cell growth could not be blocked by bicalutamide in IL-4 overexpressing LNCaP cells, but can be neutralized by bicalutamide in parental LNCaP and neo control cells. Furthermore, overexpression of IL-4 stimulates tumor growth of androgen-sensitive LNCaP cells both in intact and castrated male mice. CONCLUSIONS: Overexpression of IL-4 increases the sensitivity of androgen-sensitive LNCaP prostate cancer cells in response to androgen stimulation and enhances the growth of LNCaP cells both in the presence and absence of androgen in vitro and in vivo. These studies suggest that IL-4 plays an important role in promoting androgen-independent prostate cancer growth.

Selenium inhibition of survivin expression by preventing Sp1 binding to its promoter.

Survivin, an antiapoptotic protein highly expressed in cancer, regulates multiple cellular network associated with cancer cell viability and drug resistance. Inhibition of survivin expression has been pursued as a valid cancer therapeutic target. In this study, we showed that selenium, an effective chemopreventive agent for many types of cancers, down-regulated survivin expression. Selenium inhibited survivin expression in both mRNA and protein levels in a dose- and time-dependent manner. Using a series of survivin promoter-luciferase constructs, a 37-bp DNA element in the survivin core promoter region that mediates the ability of selenium to inhibit survivin transcription was identified. Gel mobility shift assays and chromatin immunoprecipitation analyses revealed that selenium prevents the binding of Sp1 or Sp1-like proteins to the 37-bp cis-acting DNA element in the survivin promoter. Furthermore, inhibition of survivin expression by small interfering RNA enhanced selenium's inhibitory effects on cell growth, whereas overexpression of survivin in LNCaP human prostate cancer cells desensitized cancer cells to selenium effect, suggesting that the expression of survivin plays an important role in determining the response of cancer cells to selenium. Taken together, these results suggest that selenium down-regulated survivin expression by preventing the binding of Sp1 or Sp1-like proteins to the promoter of survivin, which contributes at least in part to the inhibitory effect of selenium on survivin gene transcription. In addition, down-regulation of survivin expression may account for one of the molecular mechanisms of the anticancer effects of selenium.

LIGHT, a member of the TNF superfamily, activates Stat3 mediated by NIK pathway.

Stat3, a member of the signal transducers and activators of transcription (STAT) family, is a key signal transduction protein activated by numerous cytokines, growth factors, and oncoproteins that controls cell proliferation, differentiation, development, survival, and inflammation. Constitutive activation of Stat3 has been found frequently in a wide variety of human tumors and induces cellular transformation and tumor formation. In this study, we demonstrated that LIGHT, a member of tumor necrosis factor superfamily, activates Stat3 in cancer cells. LIGHT induces dose-dependent activation of Stat3 by phosphorylation at both the tyrosine 705 and serine 727 residues. The activation of Stat3 by LIGHT appears to be mediated by NIK phosphorylation. Expression of a kinase-inactive NIK mutant abolished LIGHT induced Stat3 activation. Overexpression of an active NIK induces Stat3 activation by phosphorylation at the both tyrosine 705 and serine 727 residues. Activation of Stat3 by NIK requires NIK kinase activity as showed by kinase assays. In addition, LIGHT increases the expression of Stat3 target genes including cyclin D1, survivin, and Bcl-xL, and stimulates human LNCaP prostate cancer cell growth in vitro which can be blocked by expression of a dominant-negative Stat3 mutant. Taken together, these results indicate that in addition to activating NF-kappaB/p52, LIGHT also activates Stat3. Activation of Stat3 together with activating non-canonical NF-kappaB/p52 signaling by LIGHT may maximize its effects on cellular proliferation, survival, and inflammation.

Interleukin-6 undergoes transition from growth inhibitor associated with neuroendocrine differentiation to stimulator accompanied by androgen receptor activation during LNCaP prostate cancer cell progression.

BACKGROUND: Interleukin-6 (IL-6) has been implicated in the modulation of growth and differentiation in many cancers, and is associated with poor prognosis in renal cell carcinoma, ovarian cancer, lymphoma, melanoma, and prostate cancer. The effects of IL-6 on the growth of LNCaP human prostate cancer cells are puzzling with some groups showing growth stimulation, while others showing growth inhibition. In this study, we investigated the discrepancy of the effects of IL-6 on prostate cancer cells. METHODS: Series of lower and higher passages of LNCaP cell sublines were generated by a long-term exposure of LNCaP cells in IL-6-containing culture media. The characteristics of these cell sublines were analyzed and the potential roles of neuroendocrine (NE) differentiation and androgen receptor (AR) activation were examined. RESULTS: We demonstrated that while short-term treatment of IL-6 inhibits LNCaP cell growth by a paracrine mechanism associated with NE differentiation, long-term treatment of IL-6 promotes LNCaP cell growth by an autocrine mechanism accompanied by an activation of AR signaling. In the lower passages (less than 28 passages) of LNCaP cells treated with IL-6, the cell growth was severely retarded which is associated with NE-like morphology and increased expression of NE markers such as neuronspecific enolase (NSE) and chromgranin A (ChgA), and loss of AR expression. However, in the higher passages (higher than 42 passages) of LNCaP cells treated with IL-6, cells started to express endogenous IL-6. At the same time, NE characteristics were disappeared, AR signaling was activated and cells growth was accelerated. Knocking down the AR activation of the higher passages of LNCaP cells abolished autocrine IL-6-induced growth stimulation. CONCLUSIONS: These studies suggest that acquisition of endogenous IL-6 production after prolong exposure of prostate cancer cells to IL-6 may contribute to an autocrine cell growth stimulation. Furthermore, the transition of IL-6 from a paracrine growth inhibitor to an autocrine growth stimulator suggests that IL-6 plays an important role during prostate cancer progression, possibly androgen-independent progression.

Mechanisms of selenium down-regulation of androgen receptor signaling in prostate cancer.

Prevention trials showed that selenium reduced prostate cancer incidence by 50%, establishing selenium as a promising chemopreventive agent for prostate cancer. Selenium inhibited human prostate cancer cell growth, blocked cell cycle progression at multiple transition points, and induced apoptotic cell death. Previous studies showed a novel mechanism of selenium anticancer action in which selenium markedly reduces androgen signaling and androgen receptor (AR)-mediated gene expression, including prostate-specific antigen (PSA), in human prostate cancer cells. The molecular mechanisms of selenium-mediated down-regulation of AR signaling are not clear. In this study, a systemic approach was taken to examine the modification of androgen signaling by selenium in human prostate cancer cells. In addition to reduced AR mRNA expression, selenium was found to initially increase the stability of AR mRNA within 6 hours while decreasing the stability of AR mRNA after 8 hours. Selenium increased AR protein degradation and reduced AR nuclear localization. Scatchard analysis indicated that selenium did not affect ligand binding to AR in LNCaP cells. Chromatin immunoprecipitation analyses showed that DHT increased the recruitment of AR and coactivators, such as SRC-1 and TIF-2, to the promoter of the PSA gene, and that recruitment was greatly diminished in the presence of 5 micromol/L selenium. On the other hand, selenium enhanced the recruitment of corepressors, such as SMRT, to the promoter of the PSA gene. Taken together, these results suggest that selenium disrupts AR signaling at multiple stages, including AR mRNA expression, mRNA stability, protein degradation, nuclear translocation, and recruitment of coregulators.