Melanoma is one of the fastest growing types of cancer worldwide in terms of incidence. To date, reports show over 92,000 new cases in the United States in 2018. Previously, we introduced protein kinase C-iota (PKC-ι) as an oncogene in melanoma. PKC-ι promotes survival and cancer progression along with PKC-zeta(ζ). In addition, we reported that PKC-ι induced metastasis of melanoma cells by increasing Vimentin dynamics. Our previous results showed that PKC-ι inhibition downregulated epithelial-mesenchymal transition (EMT), while inducing apoptosis. In this chapter, we summarized these findings which were based on the in-vitro applications of five specific atypical PKC (aPKC) inhibitors. In addition, the underlying mechanisms of the transcriptional regulation of PRKCI gene expression in melanoma is also discussed. Results demonstrated that c-Jun promotes PRKCI expression along with Interleukin (IL)-6/8. Furthermore, forkhead box protein O1 (FOXO1) acts as a downregulator of PRKCI expression upon stimulation of IL-17E and intercellular adhesion molecule 1 (ICAM-1) in melanoma cells. Overall, the chapter summarizes the importance of PKC-ι/ζ in the progression of melanoma and discusses the cellular signaling pathways that are altered upon inhibitor applications. Finally, we established that aPKCs are effective novel biomarkers for use in the design of novel targeted therapeutics for melanoma.
- PKC-iota (ι)
- PKC-zeta (ζ)
The protein kinase C (PKC) is a family of Ser/Thr kinases which are involved in transmembrane signal transduction pathways triggered by various extra and intracellular stimuli . Over time, more information has become available since the 1st discovery of PKCs in 1970s. Activation of PKCs may depend on calcium ions and cofactors like the lipid metabolite diacylglycerol (DAG) and phosphatidylserine (PS) [2, 3]. The PKC family consists of fifteen isozymes which are grouped into three on the basis of their co-factor requirements [4, 5]. First group is the conventional PKCs (cPKC) which includes the isoforms alpha (α), beta I (βI), beta II (βII) and gamma (γ) and they require calcium ions, DAG and phospholipids for the activation. Second group is the novel PKCs (nPKC) and it includes delta (δ), epsilon (ε), eta (η) and theta (θ). These are calcium ion independent but dependent on DAG and phospholipids. The aPKC isozymes are the third group, which are independent of Calcium and DAG for their activation. PKC-ζ and PKC-ι in humans (lambda (λ) is the mouse homologs of iota) are the three aPKCs. Protein kinase D, mu (μ) and some PKC-related kinases (PRK1, PRK2 and PRK3), known as PKN are also considered as PKCs .
PKCs have extremely conserved carboxyl-terminal catalytic domain (kinase domain) and PKC isozymes differ from each other on the basis of their amino-terminal (N-terminal) regulatory domain. The N-terminal domain is very important for secondary messenger binding, recruiting the enzyme to the membrane and protein-protein interactions . The pseudosubstrate (PS) domain is located at the N-terminal. PS has a peptide-sequence similar to that of a substrate but lacks alanine in the phosphoacceptor position. In the inactive form of PKCs, the PS prevents complete activation of PKC by blocking the substrate binding pocket . The PS is released upon activation [8, 9]. The activation of PKCs typically involves a cascade of three coordinated phosphorylation events [10, 11]. First, phosphorylation takes place at the activation loop triggered by phosphoinositide-dependent kinase-1 (PDK-1) [12, 13, 14]. This initiates a chain reaction that involves autophosphorylation at the turn motif that further stimulates the autophosphorylation at hydrophobic motif of N-terminal . The autophosphorylation at hydrophobic motif is the third and concluding step of the activation.
Atypical PKCs contains two structurally and functionally distinct isozymes in human, PKC-ι and PKC-ζ. The amino acid sequences in both PKC-ι and PKC-ζ are very similar to each other [15, 16]. PKC-ι is encoded by the PRKCI gene and PKC-ζ is encoded by the PRKCZ gene. They are believed to be involved in cell cycle progression, tumorigenesis, cell survival and cell migration of carcinoma cells. Additionally, aPKCs play important roles in insulin-stimulated glucose transport [16, 17]. PKC-ι specifically has a strong influence on cell cycle progression. It is also involved in changing cell polarity during cell division . Lung cancer cell proliferation is highly dependent on the PKC-ι level since it increases tumor cell proliferation by activating the ERK1 pathway . PKC-ι and PKC-ζ are expressed in both transformed and malignant melanoma . Overexpression of PKC-ι plays an important role in the chemoresistance of leukemia . PKC-ι is involved in glioma cell proliferation by regulating by phosphorylation of cyclin-dependent kinase activating kinase/cyclin-dependent kinase 7 pathway [20, 21]. A very important study by Selzer et al., investigated the presence of 11 PKC isoforms in 8 different melanoma metastases, 3 normal melanocyte cell lines and 3 spontaneously transformed melanocytes along with several melanoma tumor samples. PKC-ζ and PKC-ι were found in all transformed melanocytes and melanoma metastases samples in very high levels. PKC-ζ was also found in normal melanocytes in low levels. Figure 1 demonstrates a comparison of the aPKC expression in two normal melanocyte cell lines (PCS-200-013 and MEL-F-NEO) against two melanoma cell lines (SK-MEL-2 and MeWo) which were used for our studies in Acevedo-Duncan’s laboratory at the University of South Florida. As demonstrated in Figure 1, normal melanocytes did not show detectable levels of PKC-ι compared to the larger expression observed in SK-MEL-2 and MeWo cell lines. Moreover, PKC-ζ expression was very low in both normal melanocyte cell lines compared to heightened expression in melanoma cells. These results supported the expression patterns demonstrated by patient samples as described in Selzer et al. . All these results indicate a strong relationship between aPKCs and melanoma progression. Here, we discuss our key findings of our recent research on melanoma owing to its relationship with aPKCs in a detailed manner.
2. Atypical PKCs promote cell differentiation, survival of melanoma cells via NF-κB and PI3K/AKT pathways
Nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and phosphatidylinositol 3-kinase and protein kinase B (PI3K/AKT) pathways are often hyper-activated in many different cancers in order to promote cellular differentiation, growth and survival. Overexpression of aPKCs is often associated with anti-apoptotic effects in many cancers. We have published outcomes of
Our recent publications describe the
All five inhibitors were cytostatic to malignant cells rather than cytotoxic. Cells underwent growth arrest before apoptotic stimulation. Regardless, ICA-1S and ICA-1T showed a minor toxicity towards malignant melanoma cells, suggesting that all inhibitors were effective against malignant cells without harming normal cells. This is an indication that melanoma cells heavily rely on aPKCs to remain viable which was observed in some other cancers [19, 20, 26, 27]. These previous reports show that overexpression of aPKCs have an anti-apoptotic effect [15, 19, 20, 21, 28, 29]. Our two previous publications on the applications of aPKC specific inhibitors report apoptosis analysis on MeWo and SK-MEL-2 cells. The data confirmed that inhibition of aPKCs lead to induce apoptosis [22, 23]. Increase in Caspase-3, increase in poly ADP ribose polymerase (PARP) cleavage, and a decrease in B-cell lymphoma-2 (Bcl-2) all indicate apoptosis stimulation [30, 31, 32, 33]. All five inhibitors have demonstrated similar pattern on these markers. But, increase in Caspase-3 levels is not always a direct indication of inducing the apoptosis due to the tight binding of cleaved Caspase-3 with X-linked inhibitor of apoptosis protein (XIAP). XIAP undergoes auto-ubiquitylation, but this process delays apoptosis until all XIAP is removed . On the other hand, PARP is a known downstream target for Caspase-3, therefore we have also tested direct PARP and cleaved PARP levels upon inhibitor treatments. PARP cleavage increases upon inducing the apoptosis . Bcl-2 inhibits Caspase activity by preventing Cytochrome c release from the mitochondria and/or by binding to the apoptosis-activating factor (APAF-1). In our studies, PKC-ι and PKC-ζ inhibition decreased Bcl-2 levels which depicted an increase in apoptotic activity in both SK-MEL-2 and MeWo cell lines. These data confirms that aPKCs have an anti-apoptotic effect in the tested melanoma cells.
PI3K/AKT mediated NF-κB activation is a major anti-apoptotic pathway, wherein aPKCs play a role in releasing NF-κB complex to translocate to the nucleus and promote cell survival. Win et al. reported that PKC-ζ actively upregulates the activation of NF-κB nuclei translocation thereby inducing cancer cell survival in prostate cancer cells [36, 37]. In addition, PI3K stimulates IκB kinase (IKKα/β) through activation of AKT by phosphorylation at S473 or S463, which ultimately stimulates translocation of NF-κB complex into the nucleus, heightening cell survival . Phosphatase and tensin homolog (PTEN) regulates the levels of PI3K. Phosphorylation at S380 leads to the inactivation of PTEN, thereby increasing the levels of PI3K followed by enhancement in phosphorylated AKT (S473/S463). Our data indicates that inhibition of PKC-ι and PKC-ζ expressively decreased the levels of phosphorylated PTEN and phosphorylated AKT . This specifies that PKC-ζ and PKC-ι may upregulate the PI3K/AKT pathway to induce cellular survival of melanoma cells. Additionally, we tested the levels of NF-κB translocation by separating the nuclear extracts from the cell lysates and found that NF-κB levels in the nuclei decreased upon aPKC inhibition. This suggested that translocation of activated NF-κB into nuclei was blocked as a result of inhibition of aPKCs. Furthermore, we also found that aPKC inhibition increased the levels of inhibitor of kappa B (IκB) while decreasing the levels of phosphorylated IκB (S32) and phosphorylated IKKα/β (S176/180), confirming that both PKC-ι and PKC-ζ play a role in phosphorylation of IKKα/β and IκB: increased levels of IκB therefore remain bound to NF-κB complex and prevent the translocation to the nucleus to promote cell survival (Figure 2). As summarized in Figure 2, our data also demonstrate the effects of TNF-α stimulation on the expression of aPKCs . TNF-α is a cytokine, involved in the early phase of acute inflammation by activating NF-κB. TNF-α stimulation significantly increased NF-κB levels in both cytosol and nuclei. Increased NF-κB production promotes increases in total and phosphorylated aPKCs and increased the levels of Bcl-2, which enhanced melanoma cell survival. We observed amplified levels of IκB and NF-κB, which together enhanced the phosphorylation of IκB due to the augmented levels of aPKCs . On the other hand, PI3K/AKT signaling can be diminished by inhibiting aPKCs via downregulation of NF-κB. These results confirm that both PKC-ζ and PKC-ι are rooted in cellular survival via NF-κB and PI3K/AKT pathways.
3. PKC-ι promotes metastasis by promoting epithelial-mesenchymal transition (EMT) and activating Vimentin
Throughout EMT, epithelial cells lose apical-basal polarity, remodel the extra cellular matrix (ECM), rearrange the cytoskeleton, drive changes in signaling programs that control the cell shape maintenance and adapt gene expression to obtain mesenchymal phenotype, which is invasive and increases individual cell motility . EMT’s key features comprise downregulation of E-cadherin to destabilize tight junctions between cells and upregulation of genes such as Vimentin that may assist mesenchymal phenotype.
Vimentin is a very important structural protein which belongs to the family of type III intermediate filament proteins. Intermediate filaments (IFs) make up a vast network of interconnecting proteins between the plasma membrane and the nuclear envelope and convey molecular and mechanical information between the cell surface and the nucleus. IF protein expression is cell type and tissue specific. Mesenchymal cells, fibroblasts, lymphocytes and most types of tumor cells express Vimentin [40, 41]. Vimentin is essential for organizing microfilament systems, changing cell polarity, and thereby changing cellular motility. Moreover, increased Vimentin expression during EMT is a hallmark of metastasis which plays a very important role in gaining rear-to-front polarity for transforming epithelial cells. In addition to EMT, Vimentin expression is observed in cell mechanisms involved in cellular development, immune response and wound healing [22, 23, 42].
Vimentin is activated via phosphorylation. Various kinases such as; RhoA kinase, protein kinase A, PKC, Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), cyclin-dependent kinase 1 (CDK1), RAC-alpha serine/threonine-protein kinase (AKT1) and RAF proto-oncogene serine/threonine-protein kinases (Raf-1-associated kinases) have been shown to play a role in regulation of Vimentin via phosphorylation. Studies show that amino acid sites S6, S7, S8, S33, S38 (same as S39 since some literature use M as the starting amino acid of Vimentin), S55 (or S56), S71, S72, and S82 (S83) amongst others, serve as specific phosphorylation sites on the head region of Vimentin [41, 43, 44, 45, 46, 47, 48, 49, 50].
Our previous reports demonstrated the effects of aPKC inhibition on melanoma cell migration and invasion [22, 23]. Migration and invasion studies in cancer research are very important because the main cause of death in cancer patients is related to metastatic progression. For cancer cells to spread and distribute throughout the body, they must migrate and invade through ECM, undergo intravasation into blood stream and extravasation to form distant tumors . ACPD and DNDA treated samples demonstrated a reduction of melanoma motility but it was not conclusive which aPKC is responsible for upregulating metastasis, since both ACPD and DNDA inhibit PKC-ι and PKC-ζ . This was solved using specific PKC-ι inhibitors (ICA-1S and ICA-1T) and a PKC-ζ specific inhibitor ζ-Stat. Migration and invasion were markedly reduced for samples treated with ICA-1T and ICA-1S compared to ζ-Stat treated samples, suggesting that PKC-ι inhibition significantly diminishes melanoma cell migration and invasion suggesting that only PKC-ι is involved in EMT in melanoma . aPKC/Par6 signaling is known to stimulate EMT upon activation of TGF-β receptors in lung cancer cells. TGF-β activated aPKC/Par6 stimulates degradation of RhoA which leads to the depolymerization of filamentous actin (F-actin) and loss of epithelial structural integrity resulting a reduction in cell-cell adhesion . RhoA is a GTPase, which promotes actin stress fiber formation thereby maintains cell integrity. Furthermore, TGFβ upregulates Zinc finger protein SNAI1 (SNAIL1) and Paired related homeobox-1 (PRRX1) transcription factors that drive genetic reprogramming to facilitate EMT . Cells lose E-cadherin while gaining Vimentin during this process. We have recently reported that inhibition of PKC-ι using ICA-1T and ICA-1S significantly increased the levels of E-cadherin and RhoA while decreasing total and phosphorylated Vimentin (S39) and Par6. None of these protein levels were significantly changed as a result of PKC-ζ inhibition. We also reported that TGFβ treatments increased the expression of PKC-ι, Vimentin, phosphorylated Vimentin and Par6 while decreasing E-cadherin and RhoA . These results confirmed the involvement of PKC-ι in EMT stimulation. Immunoprecipitation of PKC-ι confirmed a strong association with Par6 in both melanoma cells which was confirmed with reverse-immunoprecipitation of Par6. Previously published reports state that both aPKCs associate with Par6 and phosphorylate at S345 . Interestingly, only PKC-ι showed an association with Par6, which confirmed that PKC-ι is a major activator of EMT in melanoma. In addition, immunoprecipitation of PKC-ι and Vimentin strongly confirmed an association between PKC-ι and Vimentin .
Phosphorylation of Vimentin at S39 is required for its activation and inhibition of PKC-ι diminishes this activation process. The reduced levels of total Vimentin observed in Western blots for ICA-1T and ICA-1S treated cells indicate that without PKC-ι, unphosphorylated Vimentin undergoes rapid degradation. In addition to activating Vimentin, PKC-ι appears to play a role in regulating Vimentin expression in some carcinoma cells .
As summarized in Figure 3, based on our published reports, we believe that TGFβ stimulated PKC-ι/Par6/RhoA and Smad2/3 pathways to induce EMT in melanoma through transcriptional activities of SNAIL1 and PRRX1. Vimentin and PKC-ι activation are upregulated simultaneously to facilitate EMT in melanoma. PKC-ι activated Vimentin thereby regulates the dynamic changes in melanoma metastasis. Our results further confirms that PKC-ι inhibition using specific inhibitors such as ICA-1T and ICA-1S, not only reduce melanoma cell survival but also negatively affects the melanoma metastatic progression by downregulating EMT. Taken together, this novel concept can be used to develop more specific effective therapeutics for melanoma patients based on PKC-ι. PKC-ι can be used as a novel biomarker to mitigate melanoma metastasis using specific inhibitors.
4. Self-regulation of PKC-ι is a crucial mechanism making PKC-ι an important novel target in melanoma anti-cancer therapeutics
In our previous study, we identified PKC-ι as a major component responsible for inducing cell growth, differentiation, survival and EMT promotion in melanoma, as a result of PKC-ι specific inhibitor applications [22, 23]. In addition to these findings, we noted that the inhibition of PKC-ι leads to a decrease in its own expression of PRKCI gene. This indicates that PKC-ι plays a role in its expression in melanoma. The PRKCI gene is located on chromosome 3 (3q26.2), a region identified as an amplicon . Our latest published results describe the transcriptional regulation of PRKCI with an insight view of cell signaling crosstalk in melanoma cells. FOXO1 and c-Jun were identified as possible transcription factors that can bind to the PRKCI promoter region through PROMO and Genomatix Matinspector. These two transcription factors (TFs) were systematically silenced to analyze the downstream effect on PKC-ι expression.
c-Jun is the first discovered oncogenic TF that is associated with metastatic breast cancer, non-small cell lung cancer and several other types of cancer [57, 58, 59]. We found a positive correlation between c-Jun with PKC-ι expression. Phosphorylation at S63 and S73 by JNKs (c-Jun N-terminal kinases) activates c-Jun, thereby increasing c-Jun targeted gene transcription. c-Jun stimulates the oncogenic transformation of ‘ras’ and ‘fos’ in several type of cancers . FOXO1 is a well-known tumor suppressor and we found it suppresses the expression of oncogenic PKC-ι. FOXO1 also plays a key role in gluconeogenesis, insulin signaling and adipogenesis. AKT is known to deactivate FOXO1 by phosphorylating FOXO1 at T24, which drives FOXO1 nuclear exclusion, leading to ubiquitination [61, 62]. Therefore, the phosphorylation of FOXO1 is an indication of its downregulation. FOXO1 plays a crucial regulatory role in both the intrinsic and extrinsic pathways of apoptosis in many types of cancers, demonstrating an association between FOXO dysregulation and cancer progression [63, 64]. Additionally, upregulation of FOXO1 inhibits cancer cell proliferation, migration and tumorigenesis . Notably, FOXO1 can also be downregulated by ERK1/2 and PKC-ι, in addition to AKT . In our most recent study, we demonstrated that, due to PKC-ι inhibition, the availability of active phosphorylated PKC-ι decreases, making it ineffective at deactivating FOXO1 through phosphorylation at T24. Importantly, this is the first showing direct involvement of PKC-ι in its own expression regulation and PKC-ι inhibition that leads to continuous upregulation of FOXO1 .
As we discussed earlier in Part 2, our previous data showed that PKC-ι inhibition significantly downregulated the PI3K/AKT1 pathway, thereby suppressing the activation of AKT [22, 23]. Downregulation of NF-κB due to PKC-ι inhibition, result in downregulation of AKT. Our latest data shows that it increases total FOXO1 level, while reducing its phosphorylated levels . This confirms that NF-κB downregulation upregulates FOXO1 activity as a result of PKC-ι specific inhibition. Elevated FOXO1 negatively influenced PKC-ι expression and phosphorylation at T555. This further confirms our previous observations with PKC-ι inhibition with ICA-1T and ICA-1S, where total PKC-ι, phosphorylated PKC-ι, NF-κB activation and activated AKT (S473) were significantly reduced . These results could be due to the tight regulation of PKC-ι expression by FOXO1, which retards PRKCI from transcription. Such results confirmed that FOXO1 is a major regulator which suppresses the expression of PRKCI. Interestingly, c-Jun and phosphorylated c-Jun (S63) levels were not significantly altered as a result of NF-κB
On the other hand,
The next three paragraphs focus on more details concerning cytokine expression changes observed as a result of PKC-ι inhibition . IL-6, IL-8, IL-17E and ICAM-1 expression were significantly altered in melanoma cells upon PKC-ι knockdown . As shown by the results of both Western blot and RT-qPCR analyses, the protein levels of IL-6 and IL-8 (as well as their mRNA levels) decreased, while the levels of IL-17E and ICAM-1 increased significantly upon PKC-ι knockdown by
IL-6 contributes to the degradation of IκB-α, leading to the upregulation of NF-κB translocation. We have previously discussed that PKC-ι stimulates NF-κB translocation through IκB-α degradation . The translocation of NF-κB to the nucleus induces cell survival through the transcription of various survival factors as well as other pro-survival cytokines [69, 73, 79]. IL-8 plays a role in regulating polymorphonuclear neutrophil mobilization. In melanoma, IL-8 has been attributed to extravasation, a key step in metastasis. Studies have shown that the expression of IL-8 in melanoma is regulated via NF-κB. When NF-κB is translocated to the nucleus, IL-8 expression increases, leading to the promotion of a more favorable microenvironment for metastasis [80, 81]. Our results indicated that both IL-6 and IL-8 expression levels decreased upon diminution of PKC-ι .
Some cytokines promote anti-tumor activity by exploiting an immune response. ICAM-1 plays a key role in the immune response, including antigen recognition and lymphocyte activation [82, 83]. ICAM-1 is known for the inhibition of tumor progression through the inhibition of the PI3K/AKT pathway. Tumor cells are exposed to cytotoxic T-lymphocytes as a result of ICAM-1 . According to ovarian cancer clinical data, inhibition of ICAM-1 expression is associated with an increased risk of metastasis for the patients within the first 5 years from the point of diagnosis [82, 83]. IL-17E (IL-25) is another anti-tumor cytokine belongs to a family of cytokines known as IL-17. Treatment with recombinant active IL-17E has been shown to decrease tumor growth of melanoma and pancreatic cancer [84, 85]. The upregulation of IL-17E is linked to the increased expression of TH17 cells. T cells, such as TH17 have been implicated in the inhibition of tumor-infiltrating effector T cells. The exact mechanism of IL-17E function in the anti-tumor effect has not been studied well enough . Particularly, our most recent results indicated that ICAM-1 and IL-17E protein levels and mRNA expression increased upon PKC-ι knockdown by
In conclusion, based on the published results from Acevedo-Duncan’s laboratory and other available information, it is suggested that PKC-ι itself plays an important role in its expression in a complex signaling web through the transcriptional activation/deactivation of c-Jun and FOXO1. The retarded activity of PKC-ι due to application of specific inhibitors such as ICA-1S and ICA-1T, causes a downregulation of the NF-κB pathway and its transcriptional activity, which reduces the expression/production of IL-6 and IL-8. In addition, as a result, the activity of AKT decreases, upregulation of FOXO1 activity takes place. FOXO1 is the most important TF regulating PKC-ι expression and IL-17E and ICAM-1 cytokines seem to play a stimulatory role for FOXO1 to attenuate PKC-ι. FOXO1 negatively regulates the expression of PKC-ι, diminishing JNK activity which leads to retard c-Jun activation. IL-6 and IL-8 expression are downregulated via PKC-ι-mediated NF-κB transcriptional activity reduction. IL-6/8 attenuation leads to STAT3/5 signaling downregulation, further reducing c-Jun expression. This whole process continues and leads to the further downregulation of NF-κB, c-Jun and upregulation of FOXO1, which leads to the continuation of the depletion of PKC-ι expression. As a result of this sequence of events, the total PKC-ι level decreases in melanoma cells, which initiated as a result of PKC-ι inhibition using specific inhibitors. These results indicate that PKC-ι is being regulated in a rather complex manner, which involves itself as a key component. PKC-ι specific inhibition using ICA-1S and ICA-1T leads to a decrease in its own production, and during this process, PKC-ι inhibition also triggers multiple anti-tumor/pro-apoptotic signaling. This makes PKC-ι one of the central key points of interest to specifically target and diminish as a means of treating melanoma. The results also strongly suggest that PKC-ι is a prime novel biomarker that can be targeted to design and develop personalized and targeted therapeutics for melanoma.
5. State of atypical PKC inhibitors
We have discussed the effects of five aPKC specific inhibitors throughout this chapter. The structures of these compounds are shown in Figure 5.
Atypical PKCs were first considered as a novel therapeutic target by Stallings-Mann et al. in 2006. They screened aurothiomalate as a potent inhibitor of the interaction between PB1 domain of PKC-ι and Par6 . Half maximal inhibitory concentration (IC50) of aurothiomalate ranged from 300 nM to 100 μM and indicated that some cell lines are insensitive (i.e. H460 and A549 lung cancer cells) to the inhibitor .
Blázquez et al. tested calphostin C and chelerythrine against West Nile virus (WNV) which significantly inhibit WNV multiplication in cell culture without affecting cell viability. They report that PKCs have also been implicated in different steps during viral replication. Calphostin C and chelerythrine two wide range PKC inhibitors that target all three PKC classes. Results indicate that atypical PKCs are involved in WNV multiplication process which can be effectively retard using said inhibitors .
Kim et al. reported the application of Echinochrome A as an inducer of cardiomyocyte differentiation from mouse embryonic stem cells. Echinochrome A was extracted from sea urchins. They investigated the potential use of Echinochrome A as an aPKC specific inhibitor and found that IC50 for PKC-ι is 107 μM under
An important study by Kwiatkowski et al. identified an azaindole-based scaffold for the development of more potent and specific PKC-ι inhibitors. They described fragmented based approach an introduced a new class of potential aPKC inhibitors based on azaindole .
The authors acknowledge the generous financial contributions from the Frederick H. Leonhardt Foundation, David Tanner Foundation, Bradley Zankel Foundation, Inc., Kyrias Foundation, Brotman Foundation of California, Baker Hughes Foundation, Irving S. Cooper Family Foundation, and the Creag Foundation.
Conflict of interests
The authors declare that they have no competing interests.