Discovery of novel dihydroartemisinin-cinnamic hybrids inducing lung cancer cells apoptosis via inhibition of Akt/Bad signal pathway.

A series of dihydroartemisinin-cinnamic acid hybrids were designed, synthesized and evaluated. Most of the tested compounds showed enhanced anti-proliferative activities than artemisinin and dihydroartemisinin, among which 16 g had the superior potency with IC50 values ranging from 5.07 µM to 7.88 µM against four tested cancer cell lines. The cell cycle arrest revealed that 16 g induced A549 cell cycle arrest at G0/G1 phase via regulation of G1-related protein expression (Cdk4). Further mechanism studies reveal that 16 g induced A549 cells apoptosis via inhibiting Akt/Bad pathway. Moreover, 16 g depolarized the mitochondria membrane potentials and induced ROS generation in A549. Additionally, 16 g blocked migration of A549 cells in a concentration-dependent manner. What's more, 16 g is barely nontoxic to zebrafish embryos. Overall, the cell cycle arrest, inhibition of Akt/Bad signal pathway, ROS generation and migration blocked might explain the potent anti-proliferative activities of these compounds.

Trans-vaccenic acid inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cells via a mitochondrial-mediated apoptosis pathway.

BACKGROUND: Intake of trans fatty acids (TFAs) from partially hydrogenated vegetable oil is associated with a variety of adverse outcomes, but little is known about the health effects of ruminant trans fats. Trans-vaccenic acid (TVA) is a naturally occurring TFA found in the fat of ruminants and in human dairy products. The present study was conducted to investigate the anticancer activity and underlying mechanisms of TVA on human nasopharyngeal carcinoma (NPC) 5-8F and CNE-2 cells. METHODS: A CCK8 assay was used to determine the effect of TVA and the Mcl-1 inhibitor S63845 on the proliferation of NPC cells. Apoptosis was measured using flow cytometry. Western blotting was used to detect the protein expression levels of factors associated with Bcl-2-family protein signaling and Akt signaling. RESULTS: TVA significantly inhibited cell proliferation in a dose-dependent manner. Mechanistic investigation demonstrated that TVA significantly decreased p-Akt levels and Bad phosphorylation on Ser-136 and Ser-112. More importantly, we discovered that the Mcl-1 inhibitor S63845 synergistically sensitized NPC cells to apoptosis induction by TVA. CONCLUSION: TVA can inhibit NPC cell growth and induced apoptosis through the inhibition of Bad/Akt phosphorylation. The combined use of TVA and Mcl-1 inhibitors offers a potential advantage for nasopharyngeal cancer treatment.

Discovery of a small-molecule inhibitor of specific serine residue BAD phosphorylation.

Human BCL-2-associated death promoter (hBAD) is an apoptosis-regulatory protein mediating survival signals to carcinoma cells upon phosphorylation of Ser99, among other residues. Herein, we screened multiple small-molecule databases queried in a Laplacian-modified naive Bayesian-based cheminformatics platform and identified a Petasis reaction product as a site-specific inhibitor for hBAD phosphorylation. Based on apoptotic efficacy against mammary carcinoma cells, N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB) was identified as a potential lead compound. In vitro biochemical analyses demonstrated that NPB inhibited the phosphorylation of hBAD specifically on Ser99. NPB was observed to exert this effect independently of AKT and other kinase activities despite the demonstration of AKT-mediated BAD-Ser99 phosphorylation. Using a structure-based bioinformatics platform, we observed that NPB exhibited predicted interactions with hBAD in silico and verified the same by direct binding kinetics. NPB reduced phosphorylation of BAD-Ser99 and enhanced caspase 3/7 activity with associated loss of cell viability in various human cancer cell lines derived from mammary, endometrial, ovarian, hepatocellular, colon, prostatic, and pancreatic carcinoma. Furthermore, by use of a xenograft model, it was observed that NPB, as a single agent, markedly diminished BAD phosphorylation in tumor tissue and significantly inhibited tumor growth. Similar doses of NPB utilized in acute toxicity studies in mice did not exhibit significant effects. Hence, we report a site-specific inhibitor of BAD phosphorylation with efficacy in tumor models.

The role of AKT and FOXO3 in preventing ovarian toxicity induced by cyclophosphamide.

Cyclophosphamide (CTX) has immunosuppressive effects and has been wildly used as one anti-cancer drug in clinical. Significant toxicity has been noticed particularly in the reproductive system. CTX promotes the maturation of ovarian follicles, decreases follicular reserve, and ultimately lead to ovarian failure or even premature ovarian failure (POF). The placental extract (HPE) has been shown to have some beneficial impact on reproductive system; however, little is known regarding to the effect of HPE on protecting CTX-induced ovarian injury and the mechanism involved. Whether human placental extracts (HPE) has a protective effect on CTX-induced toxicity on ovarian was studied by using a CTX-induced ovarian injury animal model. The effects of HEP on histopathology, the number of atretic follicles, the weight of the ovary, serum hormone levels, and apoptosis in granulosa cells were studied in mice with CTX or control vehicle. Our results have demonstrated that HPE inhibited p-Rictor, reduced the expression of Bad, Bax and PPAR, and activated Akt and Foxo3a (increased their phosphorylation). Mice treated with HPE showed higher ovarian weight, lower number of atretic follicles, higher serum levels of the hormones E2 and progesterone, and lower apoptosis and serum levels of LH and FSH in granulosa cells, than that in the control animal group. Our data show that ovarian injury can be attenuated by HPE. HPE likely protects follicular granulosa cells from undergoing significant apoptosis and reduce atresia follicle formation, therefore, alleviates CTX-induced ovarian injury.

Melatonin-Aluminum Oxide-Polymethylsiloxane Complex on Apoptosis of Liver Cells in a Model of Obesity and Type 2 Diabetes Mellitus.

We studied the effects of a melatonin-aluminum oxide-polymethylsiloxane complex (complex M) on the expression of apoptosis regulators Bcl-2 and Bad in the liver of homozygous db/db BKS.Cg-Dock7m+/+Leprdb/J mice with obesity and type 2 diabetes. Complex M or placebo was administered daily through the gastric tube during weeks 8-16 of life. In mice with type 2 diabetes mellitus receiving placebo, enhanced immunohistochemical reactions for proapoptotic Bad protein and weak response for anti-apoptotic Bcl-2 protein were observed. Administration of complex M shifted the ratio of apoptosis regulators: the area of Bcl-2 expression significantly increased and against the background of reduced Bad expression area. These findings attest to antiapoptotic effect of complex M in the liver on the model of type 2 diabetes mellitus.

Cytoprotective effect of neuropeptides on cancer stem cells: vasoactive intestinal peptide-induced antiapoptotic signaling.

Cancer stem cells (CSCs) are increasingly considered to be responsible for tumor initiation, metastasis and drug resistance. The drug resistance mechanisms activated in CSCs have not been thoroughly investigated. Although neuropeptides such as vasoactive intestinal peptide (VIP) can promote tumor growth and activate antiapoptotic signaling in differentiated cancer cells, it is not known whether they can activate antiapoptotic mechanisms in CSCs. The objectives of this study are to unravel the cytoprotective effects of neuropeptides and identify antiapoptotic mechanisms activated by neuropeptides in response to anticancer drug treatment in CSCs. We enriched and purified CSCs (CD44+/high/CD24-/low or CD133+ population) from breast and prostate cancer cell lines, and demonstrated their stemness phenotype. Of the several neuropeptides tested, only VIP could protect CSCs from drug-induced apoptosis. A functional correlation was found between drug-induced apoptosis and dephosphorylation of proapoptotic Bcl2 family protein BAD. Similarly, VIP-induced cytoprotection correlated with BAD phosphorylation at Ser112 in CSCs. Using pharmacological inhibitors and dominant-negative proteins, we showed that VIP-induced cytoprotection and BAD phosphorylation are mediated via both Ras/MAPK and PKA pathways in CSCs of prostate cancer LNCaP and C4-2 cells, but only PKA signaling was involved in CSCs of DUVIPR (DU145 prostate cancer cells ectopically expressing VIP receptor) and breast cancer MCF7 cells. As each of these pathways partially control BAD phosphorylation at Ser112, both have to be inhibited to block the cytoprotective effects of VIP. Furthermore, VIP is unable to protect CSCs that express phosphorylation-deficient mutant-BAD, suggesting that BAD phosphorylation is essential. Thus, antiapoptotic signaling by VIP could be one of the drug resistance mechanisms by which CSCs escape from anticancer therapies. Our findings suggest the potential usefulness of VIP receptor inhibition to eliminate CSCs, and that targeting BAD might be an attractive strategy for development of novel therapeutics.

Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1ß-induced inflammation and apoptosis in vitro.

Achyranthes bidentate Blume (Niuxi) is often employed for treatment of arthritis in Traditional Chinese Medicine and possesses anti-inflammatory properties. Phytochemical and pharmacological studies proved the oleanane-type saponins to be the main bioactive principles. In the present study, protective effects of A. bidentata saponins (ABS) on inflammation and apoptosis in interleukine-1ß (IL-1ß)-induced chondrocytes were investigated. Rat chondrocytes were pretreated with ABS at 3 µg/mL, 10 µg/mL, and 30 µg/mL, and subsequently stimulated with IL-1ß (10 ng/mL). Methylthiazolyldiphenyl-tetrazolium bromide assay and annexin V/propidium iodide dual staining demonstrated that ABS could protect IL-1ß-induced chondrocyte injury. ABS suppressed IL-1ß-induced apoptosis by suppressing the activation of caspase-3, inhibiting levels of proapoptotic proteins Bax and Bad, decreasing p53 protein phosphorylation, and promoting the expression of antiapoptotic protein Bcl-xL and proliferating cell nuclear antigen. IL-1ß-induced inflammation and matrix degradation were also alleviated by ABS through the downregulation of the expressions of matrix metalloproteinases 3 and 9 and cyclooxygenase-2. Moreover, ABS inhibited IL-1ß-induced nuclear factor κB activation in rat chondrocytes. We demonstrated, for the first time, the protective effects of ABS on IL-1ß-stimulated chondrocytes and their molecular mechanisms. Thus, it is suggested that ABS might be a potential drug in the treatment of osteoarthritis.

Sulforaphane induces apoptosis in adipocytes via Akt/p70s6k1/Bad inhibition and ERK activation.

Sulforaphane (SFN), an isothiocyanate isolated from cruciferous vegetables, possesses anti-oxidant and anti-cancer bioactivities. Moreover, SFN exerts its pro-apoptotic effects in some cancer lines. However, the effects and mechanisms of SFN on the regulation of apoptosis of adipocytes are still unknown. In this study, we found that SFN induced significant apoptosis in 3T3-L1 adipocytes and markedly decreased the cellular lipid content. Western blot demonstrated that SFN-induced apoptosis was mediated via the mitochondrial apoptosis pathway based on increased cleavage of poly-ADP-ribose-polymerase (PARP), release of cytochrome c into the cytoplasm, and activation of caspase-3, as well as decreased Bcl-2/Bax ratio. In addition, SFN markedly decreased phosphorylation of Akt and downstream proteins, p70s6k1 and Bad, and increased phosphorylation of ERK. Therefore, our findings clarified that SFN could induce 3T3-L1 adipocyte apoptosis via down-regulation of the Akt/p70s6k1/Bad pathway and up-regulation of the ERK pathway, suggesting SFN may be a promising agent for the treatment or prevention of obesity.

Personalized prostate cancer therapy based on systems analysis of the apoptosis regulatory network.

Targeting the androgen receptor axis provides only temporary relief for advanced prostate cancer, which often evolves into androgen-independent disease. The wide variety of signaling mechanisms connected with the pathophysiology of androgen-independent prostate cancer poses both conceptual and practical challenges for the design of efficient therapies. Analysis of apoptosis regulation in prostate cancer suggests the potential value of a systems approach that integrates information on the topology of the antiapoptotic signaling network, the signal transduction pathways that inhibit apoptosis, and the expression of proteins of the Bcl2 family. This approach could be used to identify patients most likely to respond to treatments with drugs that inhibit the signaling pathways controlling apoptosis.

Epstein-Barr virus miR-BART20-5p regulates cell proliferation and apoptosis by targeting BAD.

Although Epstein-Barr virus (EBV) BamHI A rightward transcript (BART) microRNAs (miRNAs) are ubiquitously expressed in EBV-associated tumors, the role of most BART miRNAs is unclear. In this study, we showed that Bcl-2-associated death promoter (BAD) expression was significantly lower in EBV-infected AGS-EBV cells than in EBV-negative AGS cells and investigated whether BART miRNAs target BAD. Using bioinformatics analysis, five BART miRNAs showing seed match with the 3' untranslated region (3'-UTR) of BAD were selected. Of these, only miR-BART20-5p reduced BAD expression when individually transfected into AGS cells. A luciferase assay revealed that miR-BART20-5p directly targets BAD. The expression of BAD mRNA and protein was decreased by miR-BART20-5p and increased by an inhibitor of miR-BART20-5p. PE-Annexin V staining and cell proliferation assays showed that miR-BART20-5p reduced apoptosis and enhanced cell growth. Furthermore, miR-BART20-5p increased chemoresistance to 5-fluorouracil and docetaxel. Our data suggest that miR-BART20-5p contributes to tumorigenesis of EBV-associated gastric carcinoma by directly targeting the 3'-UTR of BAD.

BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion.

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of ß-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling.

Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease.

CML cells expressing the TEL/MDS1/EVI1 fusion are resistant to imatinib-induced apoptosis through inhibition of BAD, but are resensitized with ABT-737.

Chronic myeloid leukemia is the first disease in which the potential of molecular targeted therapy with tyrosine kinase inhibitors (TKIs) was realized. Despite this success, a proportion of patients, particularly with advanced disease, are, or become, resistant to this treatment. Overcoming resistance and uncovering the underlying mechanisms is vital for further improvement of clinical outcomes. Here we report the identification, development, and characterization of a novel chronic myeloid leukemia cell line carrying the additional chromosomal aberration t(3;12)(q26;p13) resulting in expression of the TEL/MDS1/EVI1 fusion protein, which is resistant to TKIs. Resistance to TKIs was overcome by the co-administration of the BH3-mimetic, ABT-737. In addition, application of EVI1-specific small interfering RNA decreased expression of the TEL/MDS1/EVI1 fusion, reduced resistance to imatinib, and increased sensitivity to ABT-737. Subsequent studies revealed a role for the BH3-only protein BAD, probably via a phosphoinositide 3-kinase/AKT-dependent pathway, as pharmacological inhibition of AKT could also resensitize cells to death from TKIs. These findings indicate a novel pathway of TKI resistance regulated by EVI1 proteins and provide a promising means for overcoming resistance in chronic myeloid leukemia and other hematological malignancies displaying EVI1 overexpression.

Methylseleninic acid potentiates multiple types of cancer cells to ABT-737-induced apoptosis by targeting Mcl-1 and Bad.

ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins, holds great promise to complement current cancer therapies. However many types of solid cancer cells are resistant to ABT-737. One practical approach to improve its therapeutic efficacy is to combine with the agents that can overcome such resistance to restore the sensitivity. In the present study, a second-generation selenium compound methylseleninic acid (MSeA) synergistically sensitized MDA-MB-231 human breast cancer cells, HT-29 human colon cancer cells and DU145 human prostate cancer cells to apoptosis induction by ABT-737, as evidenced by greater than additive enhancement of Annexin V/FITC positive (apoptotic) cells and activation of multiple caspases and PARP cleavage. Mechanistic investigation demonstrated that MSeA significantly decreased basal Mcl-1 expression and ABT-737-induced Mcl-1 expression. Knocking down of Mcl-1 with RNAi approach supported the functional significance of this molecular target. More importantly, we identified inactivation of Bad by phosphorylation on ser-136 and ser-112 as a novel mechanism involved in ABT-737 resistance, which can be overcome by combining with MSeA. In addition, we found that expression of Bax was required for the efficient execution of synergistic sensitization. Our findings, for the first time, provide a strong mechanistic rationale for developing MSeA as a novel sensitizing agent of ABT-737.

Arginine methylation of BCL-2 antagonist of cell death (BAD) counteracts its phosphorylation and inactivation by Akt.

Protein arginine methylation is a common posttranslational modification catalyzed by a family of the protein arginine methyltransferases (PRMTs). We have previously reported that PRMT1 methylates Forkhead box O transcription factors at two arginine residues within an Akt consensus phosphorylation motif (RxRxxS/T), and that this methylation blocks Akt-mediated phosphorylation of the transcription factors. These findings led us to hypothesize that the functional crosstalk between arginine methylation and phosphorylation could be extended to other Akt target proteins as well as Forkhead box O proteins. Here we identify BCL-2 antagonist of cell death (BAD) as an additional substrate for PRMT1 among several Akt target proteins. We show that PRMT1 specifically binds and methylates BAD at Arg-94 and Arg-96, both of which comprise the Akt consensus phosphorylation motif. Consistent with the hypothesis, PRMT1-mediated methylation of these two arginine residues inhibits Akt-mediated phosphorylation of BAD at Ser-99 in vitro and in vivo. We also demonstrate that the complex formation of BAD with 14-3-3 proteins, which occurs subsequent to Akt-mediated phosphorylation, is negatively regulated by PRMT1. Furthermore, PRMT1 knockdown prevents mitochondrial localization of BAD and its binding to the antiapoptotic BCL-X(L) protein. BAD overexpression causes an increase in apoptosis with concomitant activation of caspase-3, whereas PRMT1 knockdown significantly suppresses these apoptotic processes. Taken together, our results add a new dimension to the complexity of posttranslational BAD regulation and provide evidence that arginine methylation within an Akt consensus phosphorylation motif functions as an inhibitory modification against Akt-dependent survival signaling.

Formaldehyde induces neurotoxicity to PC12 cells involving inhibition of paraoxonase-1 expression and activity.

1. Formaldehyde (FA) has been found to cause toxicity to neurons. However, its neurotoxic mechanisms have not yet been clarified. Increasing evidence has shown that oxidative damage is one of the most critical effects of formaldehyde exposure. Paraoxonase-1 (PON-1) is a pivotal endogenous anti-oxidant. Thus, we hypothesized that FA-mediated downregulation of PON1 is associated with its neurotoxicity. 2. In the present work, we used PC12 cells to study the neurotoxicity of FA and explore whether PON-1 is implicated in FA-induced neurotoxicity. 3. We found that FA has potent cytotoxic and apoptotic effects on PC12 cells. FA induces an accumulation of intracellular reactive oxygen species along with downregulation of Bcl-2 expression, as well as increased cytochrome c release. FA significantly suppressed the expression and activity of PON-1 in PC12 cells. Furthermore, H(2)S, an endogenous anti-oxidant gas, antagonizes FA-induced cytotoxicity as well as 2-hydroxyquinoline, a specific inhibitor of PON-1, which also induces cytotoxicity to PC12 cells. 4. The results of the present study provide, for the first time, evidence that the inhibitory effect on PON-1 expression and activity is involved in the neurotoxicity of FA, and suggest a promising role of PON-1 as a novel therapeutic strategy for FA-mediated toxicity.

BDNF-Akt-Bcl2 antiapoptotic signaling pathway is compromised in the brain of autistic subjects.

Although the pathogenesis of autism is not understood, emerging evidence points to apoptotic mechanisms being involved in this disorder. However, it is not known whether apoptosis signaling is deregulated in the brain of autistic subjects. This study investigates how the apoptosis-related proteins are regulated in the autistic brain. Our studies show that Bcl2 is significantly decreased, whereas the expression of p53 is increased, in the brain of autistic subjects in comparison with age-matched controls. We also found that the expression and phosphorylation/activation of Akt kinase that regulates Bcl2 are significantly decreased in the autistic brain. The down-regulation of Akt may result from a decreased concentration of brain-derived neurotrophic factor (BDNF), the growth factor that modulates Akt activities. These results suggest that down-regulation of the BDNF-Akt-Bcl2 antiapoptotic signaling pathway in the autistic brain could be one of the underlying mechanisms responsible for the pathogenesis of autism.

Synthetic triterpenoids inhibit growth, induce apoptosis and suppress pro-survival Akt, mTOR and NF-{kappa}B signaling proteins in colorectal cancer cells.

Lack of apoptotic cell death has been implicated in malignant transformation and resistance to anticancer therapies. The promotion of apoptosis in cancer cells could potentially lead to the regression and improved prognosis of refractory colorectal cancer. Synthetic triterpenoids have shown strong antitumorigenic activity towards diverse cancer cell types, but have not been investigated for colorectal cancer. In the present study, we tested the apoptosis-inducing activity of oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its C-28 methyl ester (CDDO-Me) and C-28 imidazole (CDDO-Im) derivatives in colorectal cancer cells lines. Cell growth/viability assay (MTS) demonstrated that colorectal cancer cells are highly sensitive to CDDO-Me at concentrations of 1.25 to 10 microM. The primary mode of tumor cell destruction was apoptosis as demonstrated by the cleavage of PARP-1, activation of procaspases -3, -8, and -9 and mitochondrial depolarization. Induction of apoptosis by CDDO-Me was associated with the inhibition of pro-survival Akt, NF-kappaB and mTOR signaling proteins and NF-kappaB-regulated anti-apoptotic Bcl-2, Bcl-xL, Bad and survivin. These studies provide rationale for clinical evaluation of CDDO-Me for the treatment of advanced chemotherapy refractory colorectal cancer.

Hsp27 promotes insulin-like growth factor-I survival signaling in prostate cancer via p90Rsk-dependent phosphorylation and inactivation of BAD.

Hsp27 is highly expressed in castrate-resistant prostate cancer. Although its overexpression confers resistance to androgen ablation and chemotherapy, the mechanisms by which Hsp27 inhibits treatment-induced apoptosis are incompletely defined. Castrate-resistance often correlates with increased activity of autocrine and/or paracrine growth/survival stimulatory loops including the mitogen-activated protein kinase (MAPK) and Akt pathways and insulin-like growth factor (IGF) axis components. Because Hsp27 can be activated by both MAPK and Akt pathways, it is possible that interactions between IGF-I signaling and Hsp27 phosphoactivation function to promote castrate-resistant progression. Here, we report that Hsp27 expression and phosphorylation levels correlate with IGF-I signaling and castrate-resistant progression in human prostate cancer specimens and cell lines. IGF-I induces Hsp27 phosphorylation in a time- and dose-dependent manner via p90Rsk, which interacts directly with and phosphorylates Hsp27 in vitro and in vivo. Conversely, p90Rsk inhibition using short interfering RNA or a dominant negative mutant abolishes IGF-I-induced Hsp27 phosphorylation. Hsp27 overexpression increases IGF-I-induced phosphorylation of Erk, p90Rsk, and Akt. Conversely, Hsp27 knockdown abrogates IGF-I-induced phosphorylation of Erk, p90Rsk, and Akt, thereby destabilizing Bad/14-3-3 complexes and increasing apoptotic rates. These data elucidate the interactions between Hsp27 phosphorylation and the IGF-I receptor signaling pathway and support targeting Hsp27 as a therapeutic strategy for castrate-resistant prostate cancer.

Theaflavins target Fas/caspase-8 and Akt/pBad pathways to induce apoptosis in p53-mutated human breast cancer cells.

The most common alterations found in breast cancer are inactivation or mutation of tumor suppressor gene p53. The present study revealed that theaflavins induced p53-mutated human breast cancer cell apoptosis. Pharmacological inhibition of caspase-8 or expression of dominant-negative (Dn)-caspase-8/Fas-associated death domain (FADD) partially inhibited apoptosis, whereas caspase-9 inhibitor completely blocked the killing indicating involvement of parallel pathways that converged to mitochondria. Further studies demonstrated theaflavin-induced Fas upregulation through the activation of c-jun N-terminal kinase, Fas-FADD interaction in a Fas ligand-independent manner, caspase-8 activation and t-Bid formation. A search for the parallel pathway revealed theaflavin-induced inhibition of survival pathway, mediated by Akt deactivation and Bcl-xL/Bcl-2-associated death promoter dephosphorylation. These well-defined routes of growth control converged to a common process of mitochondrial transmembrane potential loss, cytochrome c release and activation of the executioner caspase-9 and -3. Overexpression of either constitutively active myristylated-Akt (Myr-Akt) or Dn-caspase-8 partially blocked theaflavin-induced mitochondrial permeability transition and apoptosis of p53-mutated cells, whereas cotransfection of Myr-Akt and Dn-caspase-8 completely abolished theaflavin effect thereby negating the possibility of existence of third pathways. These results and other biochemical correlates established the concept that two distinct signaling pathways were regulated by theaflavins to induce mitochondrial death cascade, eventually culminating to apoptosis of p53-mutated human breast cancer cells that are strongly resistant to conventional therapies.