Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor.

Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a 'wound healing' assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model

Long-term administration of the mitochondria-targeted antioxidant mitoquinone mesylate fails to attenuate age-related oxidative damage or rescue the loss of muscle mass and function associated with aging of skeletal muscle

Age-related skeletal muscle dysfunction is the underlying cause of morbidity that affects up to half the population aged 80 and over. Considerable evidence indicates that oxidative damage and mitochondrial dysfunction contribute to the sarcopenic phenotype that occurs with aging. To examine this, we administered the mitochondria-targeted antioxidant mitoquinone mesylate {[10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl] triphenylphosphonium; 100 μM} to wild-type C57BL/6 mice for 15 wk (from 24 to 28 mo of age) and investigated the effects on age-related loss of muscle mass and function, changes in redox homeostasis, and mitochondrial organelle integrity and function. We found that mitoquinone mesylate treatment failed to prevent age-dependent loss of skeletal muscle mass associated with myofiber atrophy or alter a variety of in situ and ex vivo muscle function analyses, including maximum isometric tetanic force, decline in force after a tetanic fatiguing protocol, and single-fiber-specific force. We also found evidence that long-term mitoquinone mesylate administration did not reduce mitochondrial reactive oxygen species or induce significant changes in muscle redox homeostasis, as assessed by changes in 4-hydroxynonenal protein adducts, protein carbonyl content, protein nitration, and DNA damage determined by the content of 8-hydroxydeoxyguanosine. Mitochondrial membrane potential, abundance, and respiration assessed in permeabilized myofibers were not significantly altered in response to mitoquinone mesylate treatment. Collectively, these findings demonstrate that long-term mitochondria-targeted mitoquinone mesylate administration failed to attenuate age-related oxidative damage in skeletal muscle of old mice or provide any protective effect in the context of muscle agin

CTA095, a novel Etk and Src dual inhibitor, induces apoptosis in prostate cancer cells and overcomes resistance to Src inhibitors.

Etk is a non-receptor tyrosine kinase, which provides a strong survival signal in human prostate cancer cells. Src, another tyrosine kinase that cross-activates with Etk, has been shown to play an important role in prostate cancer metastasis. Herein, we discovered a new class of Etk inhibitors. Within those inhibitors, CTA095 was identified as a potent Etk and Src dual inhibitor. CTA095 was found to induce autophagy as well as apoptosis in human prostate cancer cells. In addition, CTA095 inhibited HUVEC cell tube formation and "wound healing" of human prostate cancer cells, implying its role in inhibition of angiogenesis and metastasis of human prostate cancer. More interestingly, CTA095 could overcome Src inhibitor resistance in prostate cancer cells. It induces apoptosis in Src inhibitor resistant prostate cancer cells, likely through a mechanism of down regulation of Myc and BCL2. This finding indicates that simultaneously targeting Etk and Src could be a promising approach to overcome drug resistance in prostate cancer

CTA095, a Novel Etk and Src Dual Inhibitor, Induces Apoptosis in Prostate Cancer Cells and Overcomes Resistance to Src Inhibitors

<div><p>Etk is a non-receptor tyrosine kinase, which provides a strong survival signal in human prostate cancer cells. Src, another tyrosine kinase that cross-activates with Etk, has been shown to play an important role in prostate cancer metastasis. Herein, we discovered a new class of Etk inhibitors. Within those inhibitors, CTA095 was identified as a potent Etk and Src dual inhibitor. CTA095 was found to induce autophagy as well as apoptosis in human prostate cancer cells. In addition, CTA095 inhibited HUVEC cell tube formation and “wound healing” of human prostate cancer cells, implying its role in inhibition of angiogenesis and metastasis of human prostate cancer. More interestingly, CTA095 could overcome Src inhibitor resistance in prostate cancer cells. It induces apoptosis in Src inhibitor resistant prostate cancer cells, likely through a mechanism of down regulation of Myc and BCL2. This finding indicates that simultaneously targeting Etk and Src could be a promising approach to overcome drug resistance in prostate cancer.</p></div

CTA095 overcomes Src inhibitor resistance in prostate cancer cells.

<p>PC3 and PC3-AZD20 (PC3 cell resistant to 20 μM AZD0530) cells were seeded at 2000 cells/well in 96 well plates overnight. The cells were treated with AZD0530 or CTA095 at the indicated concentrations. Cell viability was measured using MTT assay after 72 h. Symbols, mean; bars, standard deviation, n = 3.</p

Molecular Modeling of CTA095-Etk binding.

<p>(A) Surface view of Etk docked to CTA095 after 20 ns of MD minimization and relaxation. R₃ and the three-ring core docked deeper between Glycine-rich loop region (cyan) and hinge region (orange), R₁ and R₂ are solvent exposed. Blue: Helix C; Red: Activation Loop; Green: DFG motif (554–556); Cyan: Glycine-rich loop; Orange; Hinge Region. (B) Cartoon representation showing predicted interactions of CTA095 with the gatekeeper Thr489, DFG (554–556) motif, and Cys496. CTA095 binding stabilizes Phe555 in ‘out’ configuration, and affects the active state salt bridge formation between Lys445 and Glu460. Blue: Helix C; Red: Activation Loop; Green: DFG motif (554–556); Cyan: Glycine-rich loop; Orange; Hinge Region. Figures were generated using PyMol.</p

Induction of autophagy in PC3 cells by CTA095.

<p>Cells were grown in 6-well plate to 50% confluence and treated with CTA095. Autophagy was visualized by GFP-LC3 “puncta” (A) and immunoblot of Endogenous LC3 isoforms (B). All experiments were carried out 24 h after treatment.</p

CTA095 as a chemo sensitizer.

<p>Growth Inhibition of CTA095 and in combination with 10 μM chloroquine (CQ), or 2 ng/ml paclitaxel (PTX) to PC3 human prostate cancer cells. Cells were seeded at 5,000 cells/well in 96-well plate overnight and pretreated with the corresponding co-treatments for 1h, then treated with 2.5 μM CTA095. The cell viability was measured using MTT assay after 72 h. Columns, mean; bars, standard deviation, n = 3.</p