Cordycepin induces apoptosis by caveolin-1-mediated JNK regulation of Foxo3a in human lung adenocarcinoma.

Forkhead transcription factor (Foxo3a) is a downstream effector of JNK-induced tumor suppression. However, it is not clear whether the caveolin-1 (CAV1)-mediated JNK/Foxo3a pathway is involved in cancer cell apoptosis. We found that cordycepin upregulates CAV1 expression, which was accompanied by JNK phosphorylation (p-JNK) and subsequent Foxo3a translocation into the nucleus, resulting in the upregulation of Bax protein expression. Furthermore, we found that CAV1 overexpression upregulated p-JNK, whereas CAV1 siRNA downregulated p-JNK. Additionally, SP600125, a specific JNK inhibitor, significantly increased Foxo3a phosphorylation, which downregulated Foxo3a translocation into the nucleus, indicating that CAV1 mediates JNK regulation of Foxo3a. Foxo3a siRNA downregulated Bax protein and attenuated A549 apoptosis, indicating that the CAV1-mediated JNK/Foxo3a pathway induces the apoptosis of A549 lung cancer cells. Cordycepin significantly decreased tumor volume in nude mice. Taken together, these results indicate that cordycepin promotes CAV1 upregulation to enhance JNK/Foxo3a signaling pathway activation, inducing apoptosis in lung cancer cells, and support its potential as a therapeutic agent for lung cancer.

A bioorthogonal approach for imaging the binding between Dasatinib and its target proteins inside living cells.

Herein, we present a simple readout of the binding between a chemical drug and its target proteins in the cytoplasm by using a two-step bioorthogonal labeling method combined with spatially-localized expression of proteins. Dasatinib was modified with trans-cyclooctene (TCO), and its cytoplasmic target kinases were expressed in intracellular compartments, such as endosomes and F-actins. After bioorthogonal labeling, the colocalization between Dasatinib and its target proteins was observed in intracellular compartments.

HOXB13-mediated suppression of p21WAF1/CIP1 regulates JNK/c-Jun signaling in prostate cancer cells.

Many prostate cancer (PCa) patients die of recurrent disease due to the emergence of hormone-independent cancer cells of which the mechanism is not fully understood. Our previous studies demonstrated that most castration- resistant prostate cancers (CRPC) overexpress the HOXB13 transcription factor to confer positive growth signals. Since HOXB13 also suppresses p21WAF1/CIP1 (p21) expression, we studied the correlation between HOXB13 and p21 in selected samples of PCa. While there was no statistically significant correlation between expression of HOXB13 and p21, HOXB13-deficient tumors had three times higher odds for expressing p21 than HOXB13-positive tumors. Moreover, CRPC showed more negative correlation than hormone-dependent PCa (HDPC). Further in vitro proliferation assay demonstrated that androgen did not affect the growth-suppressive function of p21 in androgen-dependent PCa cells, suggesting that p21 seems to override the growth-promoting function of androgen and suppression of p21 expression by HOXB13 is an important step in PCa cell survival under no androgen influence. HOXB13 also inhibited AP-1 signals via suppressed expression of JNK/c-Jun. While HOXB13 suppressed p21 expression via regulation of JNK signals, alteration of p21 expression also affected c-Jun and AP-1 activity. Taken together, overexpression of HOXB13 in CRPC is an important step in avoiding the growth-suppressive effect of p21 in a harsh condition such as an androgen-deprived environment.

Anti-proliferative effects of ginsenosides extracted from mountain ginseng on lung cancer.

OBJECTIVE: To investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer. METHODS: The inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis. RESULTS: The abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed. CONCLUSION: Major ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.

HOXB13 regulates the prostate-derived Ets factor: implications for prostate cancer cell invasion.

HOXB13 has been shown to enhance the invasive potential of breast and endometrial tumors. HOXB13 is also abundant in castration-resistant prostate tumors. To determine the invasive potential of HOXB13 in prostate tumors, highly metastatic PC3 prostate cancer cells were manipulated to express HOXB13 and/or the prostate-derived Ets factor (PDEF). The PDEF is believed to reduce the invasive potential of various tumors, including prostate tumors. To further demonstrate the functional correlation between HOXB13 and PDEF, transwell invasion and gelatin zymography assays were performed. In addition, the western blot analysis was used to demonstrate the expression of PDEF target proteins involved in cancer cell migration and invasion, MMP-9 and survivin. According to the results, HOXB13 promoted PC3 cell migration and invasion. The DNA microarray analysis demonstrated that HOXB13 significantly suppressed the expression of the PDEF. Accordingly, the expression of MMP-9 and survivin was regulated by HOXB13. In addition, HOXB13 promoted the invasive potential of PC3 cells while inhibiting the PDEF. The coexpression of HOXB13 and the PDEF led to moderate retardation of the number of invasive cells, indicating that HOXB13 functionally counteracted cell invasion by reducing PDEF expression. The western blot analysis demonstrated that HOXB13 counteracted the PDEF-mediated inhibition of the expression of PDEF target proteins such as MMP-9 and survivin. The results suggest that the HOXB13-mediated promotion of tumor cell invasion is accomplished mainly through the downregulation of PDEF expression.

Five-alpha Reductase Inhibitor Influences Expression of Androgen Receptor and HOXB13 in Human Hyperplastic Prostate Tissue

Objectives Five-alpha reductase inhibitors (5ARIs) are known as chemopreventive agents in prostate cancer with a risk of high-grade disease. This study evaluated the effects of 5ARI on androgen receptor (AR) and proteins involved in prostate cell growth such as HOXB13 expression in human prostate tissue and LNCaP prostate cancer cells. Materials and Methods We retrospectively selected 21 patients who underwent TURP between March 2007 and February 2010 for previously confirmed BPH by prostate biopsy. They were grouped into control (group 1, n = 9) and 5ARI treatment (group 2, n = 12) before TURP. AR and HOXB13 expression in prostate tissue was evaluated by immunohistochemical staining. We tested the effect of 5ARI on the expression of AR, prostate specific antigen (PSA) and HOXB13 in LNCaP cells. Cells were assessed by Western blot analysis, MTT in vitro proliferation assay, and ELISA. Results: Group 2 showed stronger reactivity for AR and HOXB13 than those of the group 1. MTT assay showed death of LNCaP cells at 25uM of 5ARI. At the same time, ELISA assay for PSA showed that 5ARI inhibited secretion of PSA in LNCaP cells. Western blot analysis showed that 5ARI did not greatly alter AR expression but it stimulated the expression of HOXB13. Conclusions These results demonstrated that 5ARI influences AR and HOXB13 expression in both LNCaP cells and human prostate tissue. In order to use 5ARI in chemoprevention of prostate cancer, we still need to clarify the influence of 5ARI in ARs and oncogenic proteins and its regulation pathway. .

Five-alpha reductase inhibitor influences expression of androgen receptor and HOXB13 in human hyperplastic prostate tissue.

OBJECTIVES: Five-alpha reductase inhibitors (5ARIs) are known as chemopreventive agents in prostate cancer with a risk of high-grade disease. This study evaluated the effects of 5ARI on androgen receptor (AR) and proteins involved in prostate cell growth such as HOXB13 expression in human prostate tissue and LNCaP prostate cancer cells. MATERIALS AND METHODS: We retrospectively selected 21 patients who underwent TURP between March 2007 and February 2010 for previously confirmed BPH by prostate biopsy. They were grouped into control (group 1, n = 9) and 5ARI treatment (group 2, n = 12) before TURP. AR and HOXB13 expression in prostate tissue was evaluated by immunohistochemical staining. We tested the effect of 5ARI on the expression of AR, prostate specific antigen (PSA) and HOXB13 in LNCaP cells. Cells were assessed by Western blot analysis, MTT in vitro proliferation assay, and ELISA. RESULTS: Group 2 showed stronger reactivity for AR and HOXB13 than those of the group 1. MTT assay showed death of LNCaP cells at 25uM of 5ARI. At the same time, ELISA assay for PSA showed that 5ARI inhibited secretion of PSA in LNCaP cells. Western blot analysis showed that 5ARI did not greatly alter AR expression but it stimulated the expression of HOXB13. CONCLUSIONS: These results demonstrated that 5ARI influences AR and HOXB13 expression in both LNCaP cells and human prostate tissue. In order to use 5ARI in chemoprevention of prostate cancer, we still need to clarify the influence of 5ARI in ARs and oncogenic proteins and its regulation pathway.

Evaluation of HOXB13 as a molecular marker of recurrent prostate cancer.

Many patients with prostate cancer have disease recurrence following surgical removal of tumors and fail to respond to androgen ablation therapy. Despite the existence of a number of clinical/pathological factors, it is not possible to predict which patients will fall into this category. The results of our previous studies demonstrated that the HOXB13 homeodomain protein plays a key role in the development of prostate cancer and the progression of this malignancy. In addition, HOXB13 has been reported to predict estrogen-resistant breast cancer tumors. The purpose of this study was to investigate whether HOXB13 could be used as a molecular marker to predict prostate cancer recurrence. To examine the role of HOXB13 as a molecular marker with clinical/pathological data, the expression of HOXB13 was compared using immunohistochemistry in 57 organ-confined prostate cancer tumors obtained by radical prostatectomy. There was no significant correlation between the expression of HOXB13 and most clinical/pathological parameters, including tumor margin, invasion, pathological stage and risk level. The HOXB13 expression levels correlated with the Gleason score and there was a positive correlation with the pre-operative prostate specific antigen (PSA) levels. Accordingly, the tumor specimens from 4 patients who ultimately had biochemical failure (PSA >0.2 ng/ml), all showed a high expression of HOXB13, while their risk levels were either intermediate or high. This is the first study to report that HOXB13, together with other clinical/pathological factors, can be used as a molecular marker to predict the progression of prostate cancer.

HOXB13 promotes androgen independent growth of LNCaP prostate cancer cells by the activation of E2F signaling.

BACKGROUND: Androgen signaling plays a critical role in the development of prostate cancer and its progression. However, androgen-independent prostate cancer cells emerge after hormone ablation therapy, resulting in significant clinical problems. We have previously demonstrated that the HOXB13 homeodomain protein functions as a prostate cancer cell growth suppressor by inhibiting androgen-mediated signals. However, the role of the HOXB13 in androgen-independent growth of prostate cancer cells remains unexplained. RESULTS: In this report, we first demonstrated that HOXB13 was highly overexpressed in hormone-refractory tumors compared to tumors without prostate-specific antigen after initial treatment. Functionally, in an androgen-free environment minimal induction of HOXB13 in LNCaP prostate cancer cells, to the level of the normal prostate, markedly promoted cell proliferation while suppression inhibited cell proliferation. The HOXB13-mediated cell growth promotion in the absence of androgen, appears to be mainly accomplished through the activation of RB-E2F signaling by inhibiting the expression of the p21waf tumor suppressor. Indeed, forced expression of HOXB13 dramatically decreased expression of p21waf; this inhibition largely affected HOXB13-mediated promotion of E2F signaling. CONCLUSIONS: Taken together, the results of this study demonstrated the presence of a novel pathway that helps understand androgen-independent survival of prostate cancer cells. These findings suggest that upregulation of HOXB13 is associated with an additive growth advantage of prostate cancer cells in the absence of or low androgen concentrations, by the regulation of p21-mediated E2F signaling.

Differential CARM1 expression in prostate and colorectal cancers.

BACKGROUND: Coactivator-associated arginine methyltransferase 1 (CARM1) functions as a transcriptional coactivator of androgen receptor (AR)-mediated signaling. Correspondingly, overexpression of CARM1 has been associated with the development of prostate cancer (PCa) and its progression to androgen-independent PCa. In our preliminary study, however, the promoting effects of CARM1, with regard to androgen-stimulated AR target gene expression were minimal. These results suggested that the AR target gene expression associated with CARM1 may result primarily from non-hormone dependent activity. The goal of this study was to confirm the pattern of expression of CARM1 in human tumors and determine the mechanism of action in CARM1 overexpressed tumors. METHODS: Tissue microarray was used to determine the pattern of expression of CARM1 in human cancers by immunohistochemistry. CARM1 expression was also evaluated in prostate and colorectal surgical specimens and the clinical records of all cases were reviewed. In addition, a reporter transcription assay using the prostate-specific antigen (PSA) promoter was used to identify the signaling pathways involved in non-hormone-mediated signal activation associated with CARM1. RESULTS: The tissue microarray showed that CARM1 was particularly overexpressed in the colorectal cancers while CARM1 expression was not prevalent in the prostate and breast cancers. Further studies using surgical specimens demonstrated that CARM1 was highly overexpressed in 75% of colorectal cancers (49 out of 65) but not in the androgen-independent PCa. In addition, CARM1's coactivating effect on the entire PSA promoter was very limited in both androgen-dependent and androgen-independent PCa cells. These results suggest that there are other factors associated with CARM1 expression in PSA regulation. Indeed, CARM1 significantly regulated both p53 and NF-kappaB target gene transcription. CONCLUSIONS: The results of this study suggest that, in addition to its role in activation of steroid receptors, CARM1 functions as a transcriptional modulator by altering the activity of many transcriptional factors, especially with regard to androgen independent PCa and colorectal cancers.

HOXB13 is co-localized with androgen receptor to suppress androgen-stimulated prostate-specific antigen expression.

During the prostate cancer (PCa) development and its progression into hormone independency, androgen receptor (AR) signals play a central role by triggering the regulation of target genes, including prostate-specific antigen. However, the regulation of these AR-mediated target genes is not fully understood. We have previously demonstrated a unique role of HOXB13 homeodomain protein as an AR repressor. Expression of HOXB13 was highly restricted to the prostate and its suppression dramatically increased hormone-activated AR transactivation, suggesting that prostate-specific HOXB13 was a highly potent transcriptional regulator. In this report, we demonstrated the action mechanism of HOXB13 as an AR repressor. HOXB13 suppressed androgen-stimulated AR activity by interacting with AR. HOXB13 did neither bind to AR responsive elements nor disturb nuclear translocation of AR in response to androgen. In PCa specimen, we also observed mutual expression pattern of HOXB13 and AR. These results suggest that HOXB13 not only serve as a DNA-bound transcription factor but play an important role as an AR-interacting repressor to modulate hormone-activated androgen receptor signals. Further extensive studies will uncover a novel mechanism for regulating AR-signaling pathway to lead to expose new role of HOXB13 as a non-DNA-binding transcriptional repressor.