Protein Kinase Cδ Is a Therapeutic Target in Malignant Melanoma with NRAS Mutation

NRAS is the second most frequently mutated gene in melanoma. Previous reports have demonstrated the sensitivity of cancer cell lines carrying KRAS mutations to apoptosis initiated by inhibition of protein kinase Cδ (PKCδ). Here, we report that PKCδ inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKCδ were designed as chimeric hybrids of two naturally occurring PKCδ inhibitors, staurosporine and rottlerin. The specific hypothesis interrogated and validated is that combining two domains of two naturally occurring PKCδ inhibitors into a chimeric or hybrid structure retains biochemical and biological activity and improves PKCδ isozyme selectivity. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKCδ, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated <i>via</i> caspase-dependent apoptosis. Following PKCδ inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKCδ inhibition effectively induced cytotoxicity in BRAF mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with BRAF inhibitors. Taken together, the present work demonstrates that inhibition of PKCδ by novel small molecule inhibitors causes caspase-dependent apoptosis mediated <i>via</i> the JNK-H2AX pathway in melanomas with NRAS mutations or BRAF inhibitor resistance

Senescent inhibition overcomes TSLP-induced airway remodeling in vitro.

<p>BEAS-2B cells with stable shp16, shp21 or both were incubated with TSLP (1.5ng/ml) for 6 h. (<b>A</b>) Cells were collected and total proteins were extracted and analyzed by western blotting. (<b>B</b>) Cells were fixed and stained with SA-β-gal (upper panel) and then positive SA-β-gal cells were quantified (<i>*p>0.05</i>, **<i>p</i> < 0.05) (low panel). (<b>C</b>) Cells were stained with BrdU (<i>*p>0.05</i>, **<i>p</i> < 0.05). (<b>D</b>) Senescent inhibition overcomes TSLP-induced cell growth inhibition in vitro. The relative cell number was detected to evaluate cell growth at different time points using MTT assays.</p

Cellular senescence is induced by TSLP stimulation <i>in</i><i>vitro</i>.

<p>(<b>A</b>) TSLP-induced p16 and p21 upregulation occurs in a TSLP dose-dependent manner in human bronchial epithelial BEAS-2B cells. BEAS-2B cells were stimulated with different doses of TSLP as indicated for 6h. Protein expressions of p16, p21 and phospho-Stat3 (Try705) were detected by western blotting. (<b>B</b>) BEAS-2B cells were stimulated by 1.5ng/ml TSLP and protein expressions of p16 and p21 were detected by western blotting. (<b>C</b>) BEAS-2B cells were stimulated with 1.5ng/ml TSLP then stained for BrdU. (<i>*p< 0.05</i>). BEAS-2B cells were stimulated with 1.5ng/ml TSLP then stained for SA-β-gal activity at 6 and 24 hours post stimulation. (<b>D</b>) upper panel: SA-β-gal staining; lower panel: quantification of SA-β-gal positive cells. (*<i>p</i> < 0.05); (<b>E</b>) Ki67 staining. (<b>F</b>) Levels of TSLP in culture media were examined by ELISA. </p

Experimental therapies with WP1066 in OVA-challenged chronic asthmatic mice.

<p>(<b>A</b>) OVA-challenge was performed in BALB/c mice as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077795#B28" target="_blank">28</a>]. WP1066 was administered by intraperitoneal injection at doses of 40mg/kg 1h before the OVA-challenge. Airway resistance was measured using increasing concentration of methacholine and assessed using the flexiVent system. Results are expressed as the mean of experiments done in triplicate ± the standard error of the mean (SEM) (<i>* p<0.05</i>, <i>**p>0.05 </i><i>vs </i><i>control</i>). (<b>B</b>) TSLP, p16, p21, Ki-67, α-SMA and collagen I protein expressions were analyzed, 200×. (<b>C</b>) Bimodal H score distribution of TSLP, p16, p21, Ki67, α-SMA and collagen I immunoperoxidase reactions are presented.</p