Evaluation of antitumor effects of VEGFR-2 inhibitor F16 in a colorectal xenograft model.

OBJECTIVES: Colorectal cancer (CRC) is the third most prevalent type of cancer in the United States. The treatment options for cancer include surgery, chemotherapy, radiation, and/or targeted therapy, which show significant improvement in overall survival. Among the various available treatments, antagonizing VEGF/VEGFR-2 pathways have shown effectiveness in limiting colorectal cancer growth and improving clinical outcomes. In this regard, we hypothesized that F16, a novel VEGFR-2 inhibitor, would control colorectal cancer growth by blocking the VEGFR-2 singling pathway in both in vitro and in vivo conditions. Therefore, the current study was aimed to analyze the efficacy of F16 on the growth of Colo 320DM cells under in vitro and in vivo conditions. RESULTS: Human RT2 profiler PCR array analysis results clearly showed that angiogenesis and anti-apoptosis-related gene expressions were significantly reduced in HUVEC cells after F16 (5 µM) treatment. In addition, Western blot results revealed that F16 attenuated the downstream signaling of the VEGFR-2 pathway in HUVEC cells by up-regulating the p53 and p21 levels and down-regulating the p-AKT and p-FAK levels. Accordingly, F16 confirmed potent cytotoxic effects against the cell viability of Colo 320DM tumors, with an IC50 value of 9.52 ± 1.49 µM. Furthermore, treatment of mice implanted with Colo 320DM xenograft tumors showed a significant reduction in tumor growth and increases in survival rate compared to controls. Immunohistochemistry analysis of tumor tissues showed a reduction in CD31 levels also in F16 treated groups. CONCLUSIONS: These results justify further evaluation of F16 as a potential new therapeutic agent for treating colorectal cancers.

Effect of the HDAC Inhibitor on Histone Acetylation and Methyltransferases in A2780 Ovarian Cancer Cells.

Background andObjective: Epigenetic modifications are believed to play a significant role in the development of cancer progression, growth, differentiation, and cell death. One of the most popular histone deacetylases inhibitors (HDACIs), suberoylanilide hydroxamic acid (SAHA), also known as Vorinostat, can directly activate p21WAF1/CIP1 gene transcription through hyperacetylation of histones by a p53 independent mechanism. In the present investigation, we evaluated the correlation between histone modifications and DNA methyltransferase enzyme levels following SAHA treatments in A2780 ovarian cancer cells. Materials and Methods: Acetylation of histones and methyltransferases levels were analyzed using RT2 profiler PCR array, immunoblotting, and immunofluorescence methods in 2D and 3D cell culture systems. Results: The inhibition of histone deacetylases (HDAC) activities by SAHA can reduce DNA methyl transferases / histone methyl transferases (DNMTs/HMTs) levels through induction of hyperacetylation of histones. Immunofluorescence analysis of cells growing in monolayers and spheroids revealed significant up-regulation of histone acetylation preceding the above-described changes. Conclusions: Our results depict an interesting interplay between histone hyperacetylation and a decrease in methyltransferase levels in ovarian cancer cells, which may have a positive impact on the overall outcomes of cancer treatment.

Molecular mechanism of C-phycocyanin induced apoptosis in LNCaP cells.

The usefulness of Marine-derived products as the source of anticancer agents has been explored for many decades. The objective of our study was to investigate the molecular mechanism by which C-PC induces apoptosis in monotherapy as well as in combination treatment with a known chemotherapeutic drug named Topotecan (TPT) using prostate cancer cells (LNCaP). To determine the intracellular mechanism of action, we analyzed the gene expression profile of C-PC treated cells using human apoptosis RT2 profiler PCR array, which indicated that C-PC was able to regulate both anti- and pro-apoptotic genes significantly. Detailed analysis revealed increases in the levels of Bax, Apaf-1 (pro-apoptotic proteins) along with the activation of the key apoptotic proteases such as caspase-8, caspase-9, and caspase-3. Similarly, analysis of anti-apoptotic proteins demonstrated a decrease in the expression of Bcl-2, Mcl-1, and survivin. Results from the whole-cell incubation studies indicated that C-PC was only binding to the plasma membrane-associated receptor proteins. LNCaP cells treated with C-PC alone and in combination with TPT showed increased expression of the death receptor FAS (also known as FAS or CD95) along with cleaved PARP, confirming its importance. Our study is significant since it is providing greater insight into the apoptotic mechanisms triggered by C-PC as well as emphasizing the involvement of FAS in mediating its effects. Furthermore, our results with combination treatments suggest that-PC could improve the anticancer effects of drugs such as TPT that are currently used for cancer treatments. In addition, use of C-PC in combination can also diminish the side effects resulting from conventional chemotherapeutic agents such as TPT.

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.

Cell Cycle Arrest and Cytotoxic Effects of SAHA and RG7388 Mediated through p21WAF1/CIP1 and p27KIP1 in Cancer Cells.

BACKGROUND AND OBJECTIVE: Alterations in gene expressions are often due to epigenetic modifications that can have a significant influence on cancer development, growth, and progression. Lately, histone deacetylase inhibitors (HDACi) such as suberoylanilide hydroxamic acid (SAHA, or vorinostat, MK0683) have been emerging as a new class of drugs with promising therapeutic benefits in controlling cancer growth and metastasis. The small molecule RG7388 (idasanutlin, R05503781) is a newly developed inhibitor that is specific for an oncogene-derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. These two drugs have shown the ability to induce p21 expression through distinct mechanisms in MCF-7 and LNCaP cells, which are reported to have wild-type TP53. Our understanding of the molecular mechanism whereby SAHA and RG7388 can induce cell cycle arrest and trigger cell death is still evolving. In this study, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 using MCF-7 and LNCaP cells. MATERIALS AND METHODS: The cytotoxicity, cell cycle arrest, and apoptosis/necroptosis effects of the SAHA and RG7388 treatments were assessed using the Trypan Blue dye exclusion (TBDE) method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence assay with DEVD-amc substrate, and immunoblotting methods. RESULTS: The RG7388 treatment was able to induce cell death by elevating p21WAF1/CIP1 through inhibition of MDM2 in LNCaP, but not in MCF-7 cells, even though there was evidence of p53 elevation. Hence, we suspect that there is some level of uncoupling of p53-mediated transcriptional induction of p21WAF1/CIP1 in MCF-7 cells. CONCLUSION: Our results from MCF-7 and LNCaP cells confirmed that SAHA and RG7388 treatments were able to induce cell death via a combination of cell cycle arrest and cytotoxic mechanisms. We speculate that our findings could lead to the development of newer treatments for breast and prostate cancers with drug combinations including HDACi.

Pro-angiogenic effects of MDM2 through HIF-1α and NF-κB mediated mechanisms in LNCaP prostate cancer cells.

Hypoxia stimulates several pathways that are critical to cancer cell growth and survival, including activation of vascular endothelial growth factor (VEGF) transcription. Overexpression of VEGF and the extent of neoangiogenesis are closely correlated with tumor development and cancer metastases. Recent studies suggest MDM2 as one of the major regulators of pro-angiogenic mechanisms. To assess the direct correlation of HIF-1α and NF-κB, and the actual mechanism of MDM2 involved in the control over VEGF transcription, we exposed the LNCaP and LNCaP-MST cells (MDM2 transfected) to hypoxia. Our experiments confirm that MDM2 activation can lead to significant decrease in the levels of p53 in MDM2 transfected LNCaP-MST cells than the wild-type LNCaP cells. The results further suggest that MDM2 can be a strong regulator of both p53 dependent and independent transcriptional activity. Similarly, an increased level of other transcription factors such as HIF-1α, P300, STAT3, pAKT and NF-κB was observed. As a point of convergence for many oncogenic signaling pathways, STAT3 is constitutively activated at high frequency in a wide diversity of cancers. Our results indicate that STAT3 can directly regulate VEGF expression that is controlled by MDM2. Furthermore, it is evident from our results that NF-κB may interfere with the transcriptional activity of p53, by downregulating its levels. On the other hand, several pro-angiogenic mechanisms, including VEGF transcription which is controlled by MDM2, seem to be mediated by NF-κB.

Differential mechanisms involved in RG-7388 and Nutlin-3 induced cell death in SJSA-1 osteosarcoma cells.

Targeted therapy is becoming the mainstay of cancer treatment due to reduced side effects and enhanced tumor attack. In the last few decades, Murine Double Minute 2 (MDM2) protein has become one of the targets for developing cancer therapies. Blocking MDM2-p53 interaction has long been considered to offer a broad range of advantages during cancer treatment. In this study, we are reporting the differential mechanism of cell death induced by the two small-molecule inhibitors, named RG-7388 and Nutlin-3, that are specific for MDM2 in SJSA-1 Osteosarcoma cells (OS). Mechanistically, RG-7388 was able to enhance the phosphorylation of Mcl-1, which appears to significantly enhance its degradation, thereby relieving the pro-apoptotic protein Bak to execute the apoptosis mechanism. It was noted that the untreated SJSA-1 cells showed an accumulation of Mcl-1 levels, which was decreased following RG-7388 and to a lesser extent by Nutlin-3 and GSK-3ß (glycogen synthase kinase 3ß) inhibitor treatments. Additionally, we noted that CHIR-99021 (GSK-3ß inhibitor) blocked the cytotoxicity exerted by RG-7388 on SJSA-1 cells by decreasing Bak levels. Since Bak is an important pro-apoptotic protein, we hypothesized that phosphorylation of Mcl-1 by GSK-3ß could negatively impact the Mcl-1/Bak dimerization and relieve Bak to trigger the loss of mitochondrial membrane potential and thereby initiates apoptosis. We also observed that inhibition of GSK-3ß mediated reduction in Bak levels had a protective effect on the mitochondrial membrane integrity, and thus, caused a significant inhibition of the caspase-3 activity and PARP cleavage. Nutlin-3, on the other hand, appears to increase the levels of Bax, leading to the inactivation of Bcl-2, consequently loss of mitochondrial membrane potential and release of Cytochrome c (Cyt c) and elevation of Apaf-1 triggering apoptosis. Thus, to the best of our knowledge, this is the first study that delineates the differences in the molecular mechanism involving two MDM2 inhibitors triggering apoptosis through parallel pathways in SJSA-1 cells. This study further opens new avenues for the use of RG-7388 in treating osteosarcomas that often becomes resistant to chemotherapy due to Bcl-2 overexpression.

Regulation of cell cycle by MDM2 in prostate cancer cells through Aurora Kinase-B and p21WAF1/CIP1 mediated pathways.

Overexpression of MDM2 oncoprotein has been detected in a large number of diverse human malignancies and has been shown to play both p53-dependent and p53-independent roles in oncogenesis. Our study was designed to explore the impact of MDM2 overexpression on the levels of various cell cycle regulatory proteins including Aurora kinase-B (AURK-B), CDC25C and CDK1, which are known to promote tumor progression and increase metastatic potential. Our data from human cell cycle RT2 profiler PCR array experiments revealed significant changes in the expression profile of genes that are involved in different phases of cell cycle regulation in LNCaP-MST (MDM2 transfected) prostate cancer cells. Our current study has demonstrated a significant increase in the expression level of AURK-B, CDC25C, Cyclin A2, Cyclin B and CDK1 in LNCaP-MST cells as compared with wild type LNCaP cells that were modulated by MDM2 specific inhibitor Nutlin-3. In fact, the expression levels of the above- mentioned proteins were significantly altered at both mRNA and protein levels after treating the cells with 20 µM Nutlin-3 for 24h. Additionally, the pro-apoptotic proteins including p53, p21, and Bax were elevated with the concomitant decrease in the key anti-apoptotic proteins following MDM2 inhibitor treatment. Also, Nutlin-3 treated cells demonstrated caspase-3 activation was observed with an in-vitro caspase-3 fluorescent assay performed with caspase 3/7 specific DEVD-amc substrate. Our results offer significant evidence towards the effectiveness of MDM2 inhibition in causing cell cycle arrest via blocking the transmission of signals through AURKB-CDK1 axis and inducing apoptosis in LNCaP-MST cancer cells. It is evident from our data that MDM2 overexpression probably is the primary cause for CDK1 up-regulation in the LNCaP-MST cells, which might have occurred possibly through activation of AURK-B. However, further studies in this direction should shed more light on the intracellular mechanisms involved in the regulation of Aurora kinase-B and CDK1 axis in MDM2 positive cancers.

Differential Mechanisms of Cell Death Induced by HDAC Inhibitor SAHA and MDM2 Inhibitor RG7388 in MCF-7 Cells.

Gene expression is often altered by epigenetic modifications that can significantly influence the growth ability and progression of cancers. SAHA (Suberoylanilide hydroxamic acid, also known as Vorinostat), a well-known Histone deacetylase (HDAC) inhibitor, can stop cancer growth and metastatic processes through epigenetic alterations. On the other hand, Letrozole is an aromatase inhibitor that can elicit strong anti-cancer effects on breast cancer through direct and indirect mechanisms. A newly developed inhibitor, RG7388 specific for an oncogene-derived protein called MDM2, is in clinical trials for the treatment of various cancers. In this paper, we performed assays to measure the effects of cell cycle arrest resulting from individual drug treatments or combination treatments with SAHA + letrozole and SAHA + RG7388, using the MCF-7 breast cancer cells. When SAHA was used individually, or in combination treatments with RG7388, a significant increase in the cytotoxic effect was obtained. Induction of cell cycle arrest by SAHA in cancer cells was evidenced by elevated p21 protein levels. In addition, SAHA treatment in MCF-7 cells showed significant up-regulation in phospho-RIP3 and MLKL levels. Our results confirmed that cell death caused by SAHA treatment was primarily through the induction of necroptosis. On the other hand, the RG7388 treatment was able to induce apoptosis by elevating BAX levels. It appears that, during combination treatments, with SAHA and RG7388, two parallel pathways might be induced simultaneously, that could lead to increased cancer cell death. SAHA appears to induce cell necroptosis in a p21-dependent manner, and RG7388 seems to induce apoptosis in a p21-independent manner, outlining differential mechanisms of cell death induction. However, further studies are needed to fully understand the intracellular mechanisms that are triggered by these two anti-cancer agents.

MDM2 Overexpression Modulates the Angiogenesis-Related Gene Expression Profile of Prostate Cancer Cells.

The Murine Double Minute 2 (MDM2) amplification or overexpression has been found in many tumors with high metastatic and angiogenic ability. Our experiments were designed to explore the impact of MDM2 overexpression, specifically on the levels of angiogenesis-related genes, which can also play a major role in tumor propagation and increase its metastatic potential. In the present study, we have used the human angiogenesis RT² profiler PCR array to compare the gene expression profile between LNCaP and LNCaP-MST (MDM2 transfected) prostate cancer cells, along with LNCaP-MST cells treated with Nutlin-3, an MDM2 specific inhibitor. As a result of the overexpression of MDM2 gene in LNCaP-MST (10.3-fold), Thrombospondin 1 (THBS1), Tumor necrosis factor alpha (TNF-α) and Matrix metallopeptidase 9 (MMP9) were also found to be significantly up-regulated while genes such as Epiregulin (EREG), Tissue inhibitor of metalloproteinases 1 (TIMP1) were down-regulated. Also, we determined the total MMP activity and MMP9 expression in LNCaP, LNCaP-MST and SJSA-1 cells. Our results indicated that MDM2 level is positively correlated with MMP activity and MMP9 secretion. Our findings offer strong supporting evidence that MDM2 can impact growth and metastatic potential of cancer cells through tilting the balance towards pro-angiogenic mechanisms.