Shraddha Desai

Involvement of PKC-ι in glioma proliferation

Abstract.  Atypical protein kinase C‐iota (PKC‐ι) protects cells against apoptosis and may play a role in cell proliferation. However, in vivo, the status and function of PKC‐ι in human normal brain tissue, gliomas, benign and malignant meningiomas as well as its in vitro status in proliferating and confluent glioma cells, remains unknown. Objectives: The objectives of our research were to determine whether expression of PKC‐ι is altered either in gliomas or in benign and malignant meningiomas, compared to normal brain. In addition, we wished to establish the expression of PKC‐ι in proliferating plus in cell cycle‐arrested glioma cell lines, as well as the relationship between PKC‐ι siRNA on PKC‐ι protein content and cell proliferation. Materials and Methods: Western blot analyses for PKC‐ι were performed on 12 normal brain biopsies, 15 benign meningiomas, three malignant meningiomas and three gliomas. Results: Results demonstrated no (n = 9) or very weak (n = 3) detection of PKC‐ι in normal brain tissue. In comparison, PKC‐ι was robustly present in the majority of the benign meningiomas. Similarly, PKC‐ι was abundant in all malignant meningiomas and gliomas. Western blotting for PKC‐ι in confluent or proliferating glioma cell lines depicted substantial quantities of PKC‐ι in proliferating T98G and U‐138MG glioma cells. In contrast, confluent cells had either 71% (T98G) or 21% (U‐138MG) less PKC‐ι than proliferating cells. T98 and U‐138 MG glioma cells treated with 100 nm PKC‐ι siRNA had lower levels of cell proliferation compared to control siRNA‐A and complete down‐regulation of PKC‐ι protein content. Conclusion: These results support the concept that presence of PKC‐ι may be required for cell proliferation to take place.

A novel PKC-ι inhibitor abrogates cell proliferation and induces apoptosis in neuroblastoma

Protein Kinase C-iota (PKC-ι), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-ι confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of this research was to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-ι inhibitor in neuroblastoma cell proliferation and apoptosis. ICA-1 specifically inhibits the activity of PKC-ι but not that of PKC-zeta (PKC-ζ), the closely related atypical PKC family member. The IC(50) for the kinase activity assay was approximately 0.1μM which is 1000 times less than that of aurothiomalate, a known PKC-ι inhibitor. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-ι is an in vitro Cdk7 kinase and the phosphorylation of Cdk7 by PKC-ι was potently inhibited by ICA-1. Furthermore, our data shows that neuroblastoma cells proliferate via a PKC-ι/Cdk7/cdk2 cell signaling pathway and ICA-1 mediates its antiproliferative effects by inhibiting this pathway. ICA-1 (0.1μM) inhibited the in vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-ι inhibitor and chemotherapeutic agent for neuroblastoma.

PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

Regulation of Cdk7 activity through a phosphatidylinositol (3)-kinase/PKC-ι-mediated signaling cascade in glioblastoma

The objective of this research was to study the potential function of protein kinase C (PKC)-ι in cell cycle progression and proliferation in glioblastoma. PKC-ι is highly overexpressed in human glioma and benign and malignant meningioma; however, little is understood about its role in regulating cell proliferation of glioblastoma. Several upstream molecular aberrations and/or loss of PTEN have been implicated to constitutively activate the phosphatidylinositol (PI) (3)-kinase pathway. PKC-ι is a targeted mediator in the PI (3)-kinase signal transduction repertoire. Results showed that PKC-ι was highly activated and overexpressed in glioma cells. PKC-ι directly associated and phosphorylated Cdk7 at T170 in a cell cycle-dependent manner, phosphorylating its downstream target, cdk2 at T160. Cdk2 has a major role in inducing G(1)-S phase progression of cells. Purified PKC-ι phosphorylated both endogenous and exogenous Cdk7. PKC-ι downregulation reduced Cdk7 and cdk2 phosphorylation following PI (3)-kinase inhibition, phosphotidylinositol-dependent kinase 1 knockdown as well as PKC-ι silencing (by siRNA treatment). It also diminished cdk2 activity. PKC-ι knockdown inhibited overall proliferation rates and induced apoptosis in glioma cells. These findings suggest that glioma cells may be proliferating through a novel PI (3)-kinase-/PKC-ι/Cdk7/cdk2-mediated pathway.

The Interruption of PKC-ι Signaling and TRAIL Combination Therapy Against Glioblastoma Cells

Glioblastoma is a highly aggressive type of brain cancer which currently has limited options for treatment. It is imperative to develop combination therapies that could cause apoptosis in glioblastoma. The aim of this study was to characterize the affect of modified ICA-1, a PKC-iota inhibitor, on the growth pattern of various glioblastoma cell lines. T98G and U87 glioblastoma cells were treated with ICA-1 alone and the absolute cell numbers of each group were determined for cell growth expansion analysis, cell viability analysis, and cell death analysis. Low dose ICA-1 treatment alone significantly inhibited cell growth expansion of high density glioblastoma cells without inducing cell death. However, the high dose ICA-1 treatment regimen provided significant apoptosis for glioblastoma cells. Furthermore, this study was conducted to use a two layer molecular level approach for treating glioblastoma cells with ICA-1 plus an apoptosis agent, tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL), to induce apoptosis in such chemo-refractory cancer cells. Following ICA-1 plus TRAIL treatment, apoptosis was detected in glioblastoma cells via the TUNEL assay and via flow cytometric analysis using Annexin-V FITC/PI. This study offers the first evidence for ICA-1 alone to inhibit glioblastoma cell proliferation as well as the novel combination of ICA-1 with TRAIL to cause robust apoptosis in a caspase-3 mediated mechanism. Furthermore, ICA-1 plus TRAIL simultaneously modulates down-regulation of PKC-iota and c-Jun.

Abstract 2952: The PKC-iota inhibitor ICA-1 combined with TRAIL for glioblastoma therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastoma multiforme is a very aggressive brain tumor. Temozolimide is the standard clinical therapy following surgery which offers a limited survival advantage. The low survival rates of glioblastoma patients require further investigation into pre-clinical combination therapy options with apoptosis abilities. One such chemotherapeutic approach is the use of the novel PKC-iota inhibitor, ICA-1, combined with rhTRAIL. T98G or U87 glioblastoma cells were treated with ICA-1 for 48h or 72h, respectively, with or without rhTRAIL in a 24-well format. Based on flow cytometric analysis, the overall apoptosis rate for the T98G cells exposed to ICA-1 plus TRAIL was the most significant with no distinction between early and late apoptosis rates. In addition, the immunofluorescence analysis showed higher T98G cell death for the combination therapy. Further flow cytometric analysis showed that ICA-1 plus TRAIL generated significantly more late apoptosis for U87 cells than any other treatment. The mechanism of action for this combinatorial therapy is currently under investigation. However, our in vitro data demonstrated that ICA-1 plus TRAIL induced 60-70% apoptosis in U87 glioblastoma cells and T98G glioblastoma cells which may offer a better survival advantage in pre-clinical animal models. Citation Format: Andrea N. McCray, Shraddha Desai, Mildred Acevedo-Duncan. The PKC-iota inhibitor ICA-1 combined with TRAIL for glioblastoma therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2952. doi:10.1158/1538-7445.AM2013-2952

Abstract 3760: A novel PKC-iota inhibitor, ICA-1 abrogates cell proliferation in breast cancer

The objective of this research was to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-iota inhibitor in breast cell proliferation. PKC-α is highly overexpressed in human breast cancer and is localized depending on the trend of pathologic type of the tumor [Hum Pathol. (2008) 6:824-831]; however, little is understood about its role in regulating cell proliferation in breast cancer. Results showed that PKC-α was highly activated and overexpressed in MD-MB-468 breast cancer cells. PKC-α directly associated and phosphorylated Cdk7, a master cell cycle regulator at T170 in a cell cycle dependent manner, phosphorylating its downstream target, cdk2 at T160. PKC-α co-localized with Cdk7 in nuclear and cytoplasmic region of MD-MB-468 cells. PKC-α downregulation reduced Cdk7 phosphorylation following ICA-1 (10µM) as well as PKC-α silencing (by siRNA treatment). PKC-α knockdown inhibited overall proliferation in breast cancer cells suggesting that breast cancer cells may be proliferating through a PKC-α/Cdk7/cdk2 mediated pathway. Hence, our results emphasize the potential of ICA-1 as a novel PKC-α inhibitor and chemotherapeutic agent for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3760. doi:1538-7445.AM2012-3760

Abstract 7: PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphotidylinositol 3-kinase pathway

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Robust expression of atypical protein kinase C-iota (PKC-ι) is a hallmark of human glioma, benign and malignant meningiomas [R. Patel, et. al. (2008) Cell Prolif. 41, 122-135]. The phosphoinositide (PI) 3-kinase pathway is frequently found to be over stimulated due to activating mutations in growth factor receptors or loss of function of the tumor suppressor PTEN. Protein kinase C-ι is often activated by the PI 3-kinase pathway and is an essential downstream targeted mediator. We investigated the role of PKC-ι in two human glial tumor derived cell lines T98G and U87MG. PKC-ι directly phosphorylated and inactivated Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family at three phospho specific residues, Ser-112, Ser-136 and Ser-155. PKC-ι was also observed to co-localize and directly associate with Bad in the cytoplasm of the cells (as shown by Immunofluorescence, Immunoprecipitation, and Western blotting). Furthermore, purified, active PKC-ι induced direct phosphorylation of Bad at all three serine residues subsequently disrupting Bad/Bcl-XL dimerization. Reduction of PKC-ι upon RNA interference exhibited a corresponding reduction in Bad phosphorylation as did Wortmannin and LY294002, known PI3-Kinase inhibitors. Depletion of PKC-ι by siRNA treatment also induced apoptosis with release of cytochrome C, caspase 3 activation and PARP cleavage. These data provide evidence for novel PI3-kinase/PKC-ι/BAD cell survival pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 7. doi:10.1158/1538-7445.AM2011-7

Abstract 2974: Regulation of cyclin dependent kinase 7 through a phosphotidylinositol 3-kinase/PKC-ι mediated cell proliferation cascade

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL PKC-ι is highly over expressed in the transformed phenotype of human glioma and benign and malignant meningioma however little is understood about its role in glioma cell proliferation. Several upstream molecular aberrations and/or loss of PTEN have been implicated that lead to constitutive activation in phosphotidylinositol (PI) 3-kinase pathway. PKC-ι is one of the important downstream targeted mediators which are often up regulated by PI3-kinase. Cyclin dependent kinase activating kinase (CAK) which comprises of cyclin dependent kinase 7 (Cdk7), cyclin H and MAT1, is the master cell regulator which phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. We studied a potential function of PKC-ι in cell cycle progression and proliferation in two human glial tumor derived cells T98G and U87MG. Results show that PKC-ι directly associate and phosphorylate Cdk7 at Thr-170, which is required for Cdk activation and G1/S cell cycle transition. Furthermore, Cdk7 phosphorylated its downstream target, cyclin dependent kinase 2 (cdk2) at Thr-160. Purified PKC-ι was also observed to phosphorylate endogenous as well as exogenous Cdk7. PKC-ι knockdown with silencing RNA (siRNA) as well as PI3-kinase inhibitors, Wortmannin and LY294002 treatment exhibited corresponding reduction in phosphorylation of Cdk7 and subsequently cdk2. Collectively, these findings suggest presence of a novel PI3-kinase/PKC-ι/Cdk7/cdk2 cell proliferation pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2974. doi:10.1158/1538-7445.AM2011-2974

Abstract LB-113: PKC-iota induces uncontrolled glioblastoma cell survival by modulating proapoptotic function of Bad

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Brain tumors are a major challenge since they destroy the most important part of the human body. The aggressive and infiltrative behavior is a hallmark of glioblastoma multiforme which makes it highly incurable. PKC-iota is reported to be highly expressed in transformed phenotype of human glioma, benign and malignant meningiomas (Cell Prolif. 41:122-35, 2008) but the signaling mechanism by which PKC-iota induces glioma cell survival remains elusive. Hence, in the current study, we investigated whether PKC-iota phosphorylates and de-activates Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family. Our results demonstrated that PKC-iota co-localizes as well as directly associates and phosphorylates Bad at Ser-112, Ser-136 and Ser-155. In addition, recombinant PKC-iota induced direct phosphorylation of exogenous as well as endogenous Bad at all three serine residues as shown by in-vitro kinase assay. Recombinant PKC-zeta (is 70% homologous to PKC-iota) and PKC-delta (known to regulate the activity of Bad in rat cardiac myocytes; Mol Cells. 24:224-231, 2007) in contrast phosphorylated Bad only at Ser-112 residue in in-vitro kinase assays. Reduction of PKC-iota upon RNA interference exhibited a corresponding reduction in Bad phosphorylation at these three sites. Conversely, inhibition of PKC-zeta and delta by their corresponding siRNA did not lead to reduction of Bad phosphorylation. Additionally, disruption of Bad/Bcl-XL heterodimerization was not observed in either of the RNA silenced samples. Thus, our data strongly supports the hypothesis that PKC-iota may function as a Bad kinase directly inhibiting its pro-apoptotic function and impeding the normal homeostasis of glioma cells thereby enhancing prolonged survival of glioma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-113.