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.

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.

Enhancement of anti-melanoma activity of a plasmid expressing HIV-1 Vpr delivered through in vivo electroporation.

The development of novel treatment strategies for the effective delivery of new therapies directed against solid tumors, particularly metastatic melanoma, are required. A novel therapeutic property has previously been discovered for the HIV-1 accessory protein viral protein R (Vpr), based on the ability of this protein to induce G(2) cell cycle arrest as well as apoptosis in various tumor cell lines. Likewise, in vivo electroporation has been utilized as an effective delivery platform for DNA plasmids expressing potentially therapeutic proteins and has been targeted to normal tissues as well as tumors. Our previous findings demonstrated that delivery of a Vpr expression plasmid (pVpr) to established subcutaneous B16.F10 melanoma tumors by in vivo electroporation yielded long-term complete tumor regression in a small percentage of mice. In this study, we modified the electroporation regimen for pVpr with the goal of enhancing the anti-tumor activity of Vpr. pVpr was injected intratumorally, on days 0, 2 and 4, into established subcutaneous B16.F10 melanoma tumors followed by in vivo electroporation. Treatment with 100 mg of pVpr plus electroporation on the modified treatment days resulted in 50% of the mice undergoing complete tumor regressions coupled with long-term survival (i.e., greater than 100 days post treatment). Additional investigations established the intratumoral expression of Vpr and induction of apoptosis for a period of at least seven days after the modified pVpr treatment regimen. This report demonstrates that the anti-tumor activity of a pVpr plus electroporation regimen against established subcutaneous B16.F10 melanoma tumors can be significantly enhanced by a modified treatment schedule. In addition, it appeared that this enhanced anti-tumor effect was correlated with prolonged Vpr expression and the associated induction of intratumoral apoptosis.

Complete regression of established subcutaneous B16 murine melanoma tumors after delivery of an HIV-1 Vpr-expressing plasmid by in vivo electroporation.

Novel therapies and delivery methods directed against malignancies such as melanoma, and particularly metastatic melanoma, are needed. The HIV-1 accessory protein Vpr (viral protein R) has previously been demonstrated to induce G2 cell cycle arrest as well as in vitro growth inhibition/killing of a number of tumor cells by apoptosis. In vivo electroporation has been utilized as an effective delivery method for pharmacologic agents and DNA plasmids that express "therapeutic" proteins and has been targeted to various tissues, including malignant tumors. For the study reported here, we hypothesized that intratumoral delivery of a Vpr expression plasmid through in vivo electroporation would induce apoptosis and growth attenuation or regression of melanoma tumors. Established subcutaneous B16.F10 melanoma tumors were injected intratumorally with a Vpr-expressing (either 25 or 100 microg) plasmid, followed by electroporation, on day 0 (i.e., when tumors had attained an appropriate size) and day 4. Treatment with 25 or 100 microg of the Vpr-expressing plasmid resulted in complete tumor regression with long-term survival in 14.3 and 7.1% of the mice, respectively. In addition, electroporative delivery of the Vpr-expressing plasmid was shown to induce apoptosis in tumors after intratumoral injection. This is the first report demonstrating the ability of Vpr, when delivered as a DNA expression plasmid with in vivo electroporation, to attenuate melanoma lesion growth and induce complete tumor regression coupled with long-term survival of mice in a highly aggressive and metastatic solid tumor model.