Activation of transcription factors nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) is frequently observed in prostate cancer and has been linked with tumor cell proliferation, invasion, metastasis, and angiogenesis. In this study, we investigated the effect of ursolic acid (UA) on NF-κB and STAT3 signaling pathways in both androgen-independent (DU145) and androgen-dependent (LNCaP) prostate cancer cell lines and also prospectively tested the hypothesis of NF-κB and STAT3 inhibition using a virtual predictive functional proteomics tumor pathway technology platform. We found that UA inhibited constitutive and TNF-α-induced activation of NF-κB in DU145 and LNCaP cells in a dose-dependent manner. The suppression was mediated through the inhibition of constitutive and TNF-α-induced IκB kinase (IKK) activation, phosphorylation of IκBα and p65 and NF-κB-dependent reporter activity. Furthermore, UA suppressed both constitutive and inducible STAT3 activation in prostate cancer cells concomitant with suppression of activation of upstream kinases (Src and JAK2) and STAT3-dependent reporter gene activity. UA also downregulated the expression of various NF-κB and STAT3 regulated gene products involved in proliferation, survival, and angiogenesis and induced apoptosis in both cells lines as evidenced by DNA fragmentation and annexin V staining. In vivo, UA (200 mg/kg b.w.) treated for 6 weeks inhibited the growth of DU145 cells in nude mice without any significant effect on body weight. Overall, our results from experimental and predictive studies suggest that UA mediates its anti-tumor effects through suppression of NF-κB and STAT3 pathways in prostate cancer.

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Purpose: Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third cause of global cancer mortality. Increasing evidence suggest that STAT3 is a critical mediator of oncogenic signaling in HCC and controls the expression of several genes involved in proliferation, survival, metastasis, and angiogenesis. Thus, the novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC.Experimental Design: The effect of butein on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation, and apoptosis was investigated. The in vivo effect of butein on the growth of human HCC xenograft tumors in male athymic nu/nu mice was also examined.Results: We tested an agent, butein, for its ability to suppress STAT3 activation in HCC cells and nude mice model along with prospectively testing the hypothesis of STAT3 inhibition in a virtual predictive functional proteomics tumor pathway technology platform. We found that butein inhibited both constitutive and inducible STAT3 activation in HCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src and Janus-activated kinase 2. Butein inhibited proliferation and significantly potentiated the apoptotic effects of paclitaxel and doxorubicin in HCC cells. When administered intraperitoneally, butein inhibited the growth of human HCC xenograft tumors in male athymic nu/nu mice.Conclusions: Overall, cumulative results from experimental and predictive studies suggest that butein exerts its antiproliferative and proapoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.

BACKGROUND AND PURPOSEActivation of pro-inflammatory transcription factors NF-kB and signal transducer and activator of transcription 3 (STAT3) is one of the major contributors to both pathogenesis and chemoresistance in multiple myeloma (MM), which results in high mortality rate. Thus, in the present study, we investigated whether celastrol could suppress the proliferation and induce chemosensitization of MM cells by interfering with NF-kB and STAT3 activation pathways. EXPERIMENTAL APPROACHThe effects of celastrol were investigated using both a virtual predictive tumour cell system and different MM cell lines resistant to doxorubicin, melphalan and bortezomib. KEY RESULTSCelastrol inhibited the proliferation of MM cell lines regardless of whether they were sensitive or resistant to bortezomib and other conventional chemotherapeutic drugs. It also synergistically enhanced the apoptotic effects of thalidomide and bortezomib. This correlated with the down-regulation of various proliferative and anti-apoptotic gene products including cyclin D1, Bcl-2, Bcl-xL, survivin, XIAP and Mcl-1. These effects of celastrol were mediated through suppression of constitutively active NF-kB induced by inhibition of IkBa kinase activation; and the phosphorylation of IkBa and of p65. Celastrol also inhibited both the constitutive and IL6-induced activation of STAT3, which induced apoptosis as indicated by an increase in the accumulation of cells in the sub-G1 phase, an increase in the expression of pro-apoptotic proteins and activation of caspase-3. CONCLUSIONS AND IMPLICATIONSThus, based on our experimental findings, we conclude that celastrol may have great potential as a treatment for MM and other haematological malignancies.

A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the ␤-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca 2؉ ) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca 2؉ -mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca 2؉ ionophore A23187, cGMP-gated channel agonist 8-bromocGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca 2؉ influx seen in the rd photoreceptors. Ca 2؉ -induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca 2؉ influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity.

Sustained elevation in cGMP and a concomitant increase in intracellular Ca(2+) levels in the rd1 photoreceptors are followed by a rapid loss of photoreceptors. In a murine-derived photoreceptor cell line, 661W, treated with the phosphodiesterase inhibitor IBMX or the cyclic GMP-gated channel agonist 8-bromo-cGMP, it was previously found that the induced cell death was mediated by calpain and caspase-3. Because oxidative stress is a common product of ionic imbalance or elevated Ca(2+), we tested the role of oxidative stress in cGMP-induced photoreceptor cell death. In the rd1 mouse retina, oxidative stress was found to precede calpain and caspase-3 activation. In 661W cells, the increase in intracellular cGMP and Ca(2+) resulted in the generation of reactive oxygen species (ROS), the activation of oxidative stress enzymes, and the activation of calpain, followed by apoptosis mediated by the effector caspase-3. All these events, including calpain activation, were ameliorated by docosahexanoic acid (DHA). The cell-permeable inhibitor of calpain, SJA6017, while inhibiting cell death, had no effect on the generation of oxidative stress. These results establish a central role for oxidative stress in cGMP-induced cell death and suggest a ROS-mediated sequential activation of signal transduction events, which provide targets for future treatment strategies.

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