HDAC Inhibitor SAHA Sensitizes Metformin-Induced Cell Death in A2780 Ovarian Cancer Cells

HDAC inhibitor SAHA sensitizes Metformin-induced cell death in A2780 ovarian cancer cells Amal Alzahrani1, Ph.D student Theodore Lemuel Mathuram2, Ph.D., Research Associate Appu Rathinavelu1,2, Ph.D., Professor, Executive Director 1College of Pharmacy, 2Rumbaugh-Goodwin Institute for Cancer Research Objective. The study was conducted to assess the efficacy of metformin in combination with SAHA in ovarian cancer cell line (A2780). Background. Ovarian cancer is the seventh most common cancer among women with the highest mortality rate. The high mortality rate of ovarian cancer is attributed to the fact that most women are diagnosed at an advanced stage with poor survival rate. Recently, many studies have confirmed a profound effect of the known anti-diabetic drug, metformin, on various types of cancer including the ovarian cancer. Our study aims to repurpose metformin, by sensitizing A2780 cells with SAHA (pan-HDAC inhibitor) at lower doses. Methods. For this study, MTT assay was conducted with A2780 cells treated with metformin or SAHA, combination of metformin and SAHA at different doses for 24, 48 h. Western Blotting analysis was conducted to assess the protein expression levels of cytochrome C and p21. Results. Combination of metformin 0.1mM and SAHA 10 µM was able to significantly reduce cell viability after 48 h compared to metformin and SAHA alone. Moreover, the combination of metformin 0.1mM and SAHA 10 µM demonstrated significant upregulation of cytochrome C and p21 levels comparing to metformin and SAHA alone. Conclusion. Our results indicate that the reduction in cell viability, and upregulation of cytochrome C and p21 levels may be due to the sensitizing effect of SAHA to metformin. Grants. This study was funded by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida

The Emerging Role of GSK-3 Inhibitors as Promising Drug Candidates in NSCLC

Objective. This study was conducted to assess the apoptotic role of GSK-3 inhibitors: BIO and CHIR 98014 against H460 K-Ras mutant (mut) and H1975 K-Ras wild type (wt) Non-small cell lung cancer (NSCLC) cells. Background. NSCLC accounts for 80% - 85% of lung cancers, with mutation of KRAS being the most frequent aberration. Our study was designed to determine the use of GSK-3 inhibitors as apoptotic inducers against NSCLC cells. Methods. In our study, the cell viability and cell proliferation of H460 and H1975 were measured using MTT and BrdU assay after 24, 48, and 72 h of BIO and CHIR 98014 treatments. Imaging studies to assess Reactive oxygen species (ROS), Mitochondrial Membrane Potential (MMP), and Caspase-3/7 cleavage were conducted. The trans-endothelial migratory assay was conducted to assess the potential of BIO and CHIR 98014 for inhibiting cancer metastasis. Western blot analysis was conducted for measuring pGSK-3, phospho-p53, p21, XIAP, BAX, LC3B, Caspase-3, and Caspase-9 levels. Results. GSK-3 inhibitors significantly reduced the cell viability after 24 h treatment in H1975 compared to H460 cells. In addition, BIO and CHIR 98014 demonstrated significant upregulation of ROS levels, while decreasing its mitochondrial membrane potential, leading to cleavage of Caspase-3, 7, and 9. Interestingly, a significant elevation of phospho-p53, p21, and LC3B levels was observed with BIO and CHIR 98014 treatments. Conclusion. Our results indicated that GSK-3 inhibitors were able to induce cell death by activating both extrinsic and intrinsic apoptotic pathways. Grants. This study was funded by the PFRDG grant 334818 and the financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida

The Neuroprotective Role of Nimodipine and nNOS Inhibitor Against Bortezomib-Induced Cell Death in Schwann Cells

Objective. The study was conducted to assess the neuroprotective role of Nimodipine (Nim) and nNOS inhibitor (nNi) against bortezomib (BTZ)-induced cell death in Schwann cells (RT4-D6P2T). Background. Schwann cells (SC) are myelinating cells, known to maintain the integrity and regeneration of neurons in the peripheral nervous system (PNS). Our study was designed to determine the molecular mechanisms involved in the use of neuroprotective agents against bortezomib-induced damage in SC. Methods. In our study, the cell viability of BTZ-treated cells was measured after 24, 48, and 72 h of treatments, followed by neuroprotective studies with Nim and nNi. Reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were assessed using DCFDA and JC-1 staining method. Western blot analysis was conducted for measuring phospho-epidermal growth factor receptor (pEGFR), myelin basic protein (MBP), Protein kinase B (AKT) and phospho-Protein kinase B (pAKT) protein levels. Results. BTZ (1 µM) treatment was able to significantly reduce the cell viability after 24 h treatment compared to untreated controls. In addition, pre-treatment with Nim (10 µM) and nNi (1 µM) demonstrated significant upregulation of pAKT levels in BTZ-treated cells, while a significant decrease in MBP expression levels was observed. Interestingly, marginal decrease was observed in Nim and nNi pre-treated cells. Conclusion. Our results indicate that the neuroprotective role of Nim and nNi may be due to upregulation of pAKT and related pathways. Grants. This study was funded by the PFRDG grant 334818 and the financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida

The apoptotic effect of GSK-3 inhibitors: BIO and CHIR 98014 on H1975 lung cancer cells through ROS generation and mitochondrial dysfunction.

OBJECTIVE: GSK-3 has been reported to be upregulated in malignant diseases, including lung cancers, thus suggesting it to be a valid target for cancer treatment. The study elucidates the possible mechanism involved in the ability of GSK-3 inhibitors: BIO and CHIR 98014 to regulate proteins involved in cell death of H1975 lung cancer cells. RESULTS: BIO and CHIR 98014 successfully induced apoptosis at lower concentrations in H1975 cells but not in H460 lung cancer cells. Moreover, increased ROS generation and depolarization of mitochondrial membrane potential were observed in both treatments. Cleavage of caspase-3 was observed in both BIO and CHIR 98014-treated cells after 72 h with monolayer and tumorsphere cell culture models. CONCLUSIONS: The use of GSK-3 inhibitors shows promising apoptotic abilities in clinical cancer treatments, particularly for lung cancer cells. This study is the first report to describe the significant apoptotic effects of BIO and CHIR 98014 through multiple mechanisms of H1975 NSCLC that are linked to their proliferative and migratory capacities

PD-L1 Expression Due to Epigenetic Modifications During Histone Deacetylase Inhibition in Cancer Cells

Objective: We analyzed the expression of PD-L1 following inhibition of HDACs (Histone Deacetylase) with SAHA (Suberoylanilide Hydroxamic Acid). Background: SAHA is a broad-spectrum HDAC Inhibitor, which plays an important role in regulating the gene expressions to induce cell cycle arrest and cell death in cancer cells. Programmed Cell Death Ligand-1 (PD-L1) can be expressed on the surface of cancer cells, which typically confers aggressive growth and immune-suppression characteristics. SAHA treatment was expected to alter PD-L1 expression in cancer cells. Methods: The HCC827 cells were treated with HDACi and used for gene expression analysis using Microarray, qRT-PCR and Western blot. Results: SAHA treatment was able to reduce the levels of PD-L1 expression in a dose and time-dependent manner. The PD-L1 level was found to be negatively correlated to the p21 expression in HCC827 cells after treatment with HDACi. Interestingly the qRT-PCR Array analysis revealed significant decreases in the mRNA levels of methyltransferases such as DNMT3A, DNMT3B, PRMT1, SUV39H1, KAT6B, NSD1, SETD1B, and WHSC1. SAHA treatment increased the levels of acetyl forms of H2B, H3, and H4, while decreasing the levels of the methyltransferases. Conclusion: HDACi treatment was leading to hyper-acetylation of histones while the levels of methyltransferases were decreased significantly. It is suspected that the decrease in the PD-L1 and methyltransferases levels are due to inhibition of transcription following treatment with SAHA. Grants: Research was supported by the Royal Dames of Cancer Research Inc., Fort Lauderdale, Florida