The interruption of atypical PKC signaling and Temozolomide combination therapy against glioblastoma.

Current treatment options of glioblastoma include chemotherapy and limited surgical resection. Temozolomide (TMZ) is the current therapeutic choice for chemotherapy. Still, it has severe limitations due to the development of resistance that occurs by genetic modification and constitutive activation of several cell signaling pathways. Therefore, it is essential to develop combination therapy of TMZ with other novel compounds to prevent the development of chemo-resistance. In this study, we used two inhibitors; ICA, an inhibitor of PKC-ι and ζ-Stat, an inhibitor of PKC-ζ. T98G and U87MG glioblastoma cells were treated with either ICA or ζ-stat or TMZ monotherapies, as well as TMZ were combined with either ICA or ζ-stat for five consecutive days. Our in vitro results exhibited that ICA when combined with TMZ, significantly decreased the viability of cancerous cells compared with untreated or TMZ or ICA monotherapies. Additionally, glioblastoma cells were remarkably undergoing apoptosis against the combination treatment of TMZ and ICA nucleotide compared with untreated control cells, as suggested by our Annexin-V/PI flow cytometric analysis. Moreover, the combination of TMZ and ICA also decreased the invasion of glioblastoma cell lines by acting on FAK/Paxillin pathway, as evidenced by scratch assay, transwell invasion assay, Western blot and immunoprecipitation analysis. Furthermore, our in vivo data presented that the combination of ICA and TMZ also reduced glioblastoma tumor growth and volume in mice. These data suggest that atypical PKCs, particularly PKC-ι might be an important therapeutic target as adjuvant therapy in the treatment of glioblastoma.

The Atypical Protein Kinase C Small Molecule Inhibitor ζ-Stat, and Its Effects on Invasion Through Decreases in PKC-ζ Protein Expression.

Ovarian cancer is estimated to reach 22,530 diagnoses and cause 13,980 cancer deaths per year. The most common histology diagnosed of ovarian cancer is epithelial ovarian carcinomas (EOC). An aggressive epithelial subtype is clear cell ovarian carcinoma (CCOC) and is characterized as a non-serous ovarian cancer. Protein kinase C (PKC) is an enzymatic family of proteins that have been found to be a component in cancer progression, tissue invasion, and metastasis. The atypical PKC (aPKC) isoforms, PKC-ι and PKC-ζ, have been suggested to participate in the increased proliferation of ovarian cancers. Previous studies have indicated that novel aPKC inhibitors ICA-1S and ζ-Stat decreased the migratory behaviors of colorectal cancer cells and were selective for PKC-ι/λ and PKC-ζ, respectively. The aims of this investigation were to further determine the binding mechanisms of ζ-Stat, expand on the tissue range of these compounds, investigate the therapeutic potential of ζ-Stat in CCOC, and to illustrate the disruption of invasion via the PKC-ζ signaling cascade. The methods utilized were molecular docking and virtual target screening, Western blot analysis, end-point PCR, GST pull down, cell viability and invasion and migration assays. We discovered that the small molecule inhibitor, ζ-Stat, is a prospective drug candidate to investigate as a novel potential treatment for CCOC. We also found that the PKC-ζ/Ect2/Rac1 activation pathway was decreased by ζ-Stat, which in turn decreased invasive behavior of CCOC.

Atypical Protein Kinase-C inhibitors exhibit a synergistic effect in facilitating DNA damaging effect of 5-fluorouracil in colorectal cancer cells.

Colorectal cancer (CRC) is the third most common malignancy and the fourth most common cause of cancer-related death worldwide. The treatment of metastatic CRC considered palliative for many years aiming for an improved life, with little hope of a cure, highlighting the need for developing novel targeted therapy for CRC. Human protein kinases constitute a complicated system with complex internal and external interactions, which stimulates various cellular processes such as cell growth, metabolism, survival, and apoptosis. This study investigated the effect of a combination of atypical Protein Kinase C (PKC) inhibitor (either ICA-I or ζ-Stat) and 5-FU (a thymidylate synthase inhibitor) on CRC cells viability concerning cellular DNA damage. In this study, we took multiple approaches such as colony formation assay, flow cytometry, DNA ladder assay, TUNEL assay, etc. to examine the CRC cell viability and apoptosis as a function of combination treatment. Our findings showed that the combination of atypical PKC inhibitor and 5-FU synergistically reduced the viability of CRC cells and induced apoptosis. Additionally, the DNA ladder and TUNEL assays indicated that there was a notable DNA damage and fragmentation because of lack of thymidylate synthase and due to the deactivation of atypical PKC dependent CDK7. These data suggest that the simultaneous knockdown of upstream atypical PKC protein and downstream DNA damage repairing mechanism would be a useful approach to combat CRC and to improve overall patients' survival rate.

Analysis of PKC-ζ protein levels in normal and malignant breast tissue subtypes.

It is estimated that breast cancer will be the second leading cause of cancer-associated mortality in women in 2018. Previous research has demonstrated that the atypical protein kinase C-ζ (PKC-ζ) is a component of numerous dysregulated pathways in breast cancer, including cellular proliferation, survival, and cell cycle upregulation. The present study investigated the PKC-ζ protein in breast tissue to evaluate its potential as a biomarker for breast cancer invasion, and demonstrated that an overexpression of PKC-ζ protein can be indicative of carcinogenesis. The present study analyzed the expression of PKC-ζ in individuals with no tumor complications and malignant female human breast tissue samples (lobular carcinoma in situ, invasive lobular carcinoma, ductal carcinoma in situ and invasive ductal carcinoma) with the use of western blot analysis, immunohistochemistry and statistical analysis (83 samples). The present study also evaluated the invasive behavior of MDA-MB-231 breast cancer cells following the knockdown of PKC-ζ with a Transwell invasion assay and an immunofluorescent probe for filamentous actin (F-actin) organization. The data demonstrated that PKC-ζ expression was identified to be higher in invading tissues when compared with non-invading tissues. The results also suggest that PKC-ζ is more abundant in ductal tissues when compared with lobular tissues. In addition, the protein studies also suggest that PKC-ζ is a component for invasive behavior through the Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras homolog gene family member A (RhoA) pathway, and PKC-ζ is required for the F-actin reorganization in invasive cells. Therefore, PKC-ζ should be considered to be a biomarker in the development of breast cancer as well as an indicator of invading tumor cells.

The modulation of actin dynamics via atypical Protein Kinase-C activated Cofilin regulates metastasis of colorectal cancer cells.

Colorectal cancer (CRC) is the third most common cancer in the United States. The exact mechanism of CRC cells metastasis is poorly understood. Actin polymerization is thought to be an initial step in the cancer cell motility cycle which drives the formation of cell protrusions and defines the direction of migration. Cofilin, a significant actin-regulating molecule, regulates the migration of cancer cells by the formation of lamellipodia and filopodia, however, little is known about the upstream regulation of cofilin. In this study, the effect of atypical Protein Kinase C (atypical PKC) on Cofilin activity in CRC was studied. This study demonstrates that the atypical PKC inhibition impedes the metastasis of CRC cells by increasing phospho-Cofilin (S3) and changing actin organization.

Protein Kinase C-ζ stimulates colorectal cancer cell carcinogenesis via PKC-ζ/Rac1/Pak1/ß-Catenin signaling cascade.

Colorectal cancer (CRC) is the second most common cancer in the world and death from CRC accounts for 8% of all cancer deaths both in men and women in the United States. CRC is life-threatening disease due to therapy resistant cancerous cells. The exact mechanisms of cell growth, survival, metastasis and inter & intracellular signaling pathways involved in CRC is still a significant challenge. Hence, investigating the signaling pathways that lead to colon carcinogenesis may give insight into the therapeutic target. In this study, the role of atypical Protein Kinase C (aPKC) on CRC was investigated by using two inhibitors of that protein class: 1) ζ-Stat (8-hydroxynaphthalene-1,3,6-trisulfonic acid) is a specific inhibitor of PKC-ζ and 2) ICA-I 5-amino-1-(2,3-dihydroxy-4-hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide) is a specific inhibitor of PKC-ι. The cell lines tested were CCD18CO normal colon epithelial and LOVO metastatic CRC cells. The inhibition of aPKCs did not bring any significant toxicity on CCD18CO normal colon cell line. Although PKC-ι is an oncogene in many cancers, we found the overexpression of PKC-ζ and its direct association with Rac1. Our findings suggest that the PKC-ζ may be responsible for the abnormal growth, proliferation, and migration of metastatic LOVO colon cancer cells via PKC-ζ/Rac1/Pak1/ß-Catenin pathway. These results suggest the possibility of utilizing PKC-ζ inhibitor to block CRC cells growth, proliferation, and metastasis.