Belal Al-Husein

Antiangiogenic Therapy for Cancer: An Update

The idea of antiangiogenic therapy was the brainchild of Dr. Judah Folkman in the early 1970s. He proposed that by cutting off the blood supply, cancer cells would be deprived of nutrients and, hence, treated. His efforts paid off when bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor, was approved as antiangiogenic therapy in 2004 for the treatment of colon cancer. Since then, an array of antiangiogenic inhibitors, either as monotherapy or in combination with other cytotoxic and chemotherapy drugs, have been developed, used in clinical trials, and approved for the treatment of cancer. Despite this important breakthrough, antiangiogenic therapy for cancer met with a number of hurdles on its way to becoming an option for cancer therapy. In this article, we summarize the most current information on the mechanisms of tumor angiogenesis, proangiogenic and antiangiogenic factors, potential targets and their mechanisms of action, and experimental evidences, as well as the most recent clinical trial data on antiangiogenic agents for cancer therapy.

Anticancer Efficacy of Simvastatin on Prostate Cancer Cells and Tumor Xenografts Is Associated with Inhibition of Akt and Reduced Prostate-Specific Antigen Expression

Prostate cancer is the second-leading cause of cancer-associated death among men in the United States. There has been renewed interest in the potential therapeutic benefits of statins for cancer. Simvastatin, a widely used generic drug for preventing cardiovascular events, is well known for its effects on cellular proliferation and inflammation, two key processes that also determine the rate of tumor growth. Although a growing body of evidence suggests that statins have the potential to reduce the risk of many cancers, there are discrepancies over the pro- and anticancer effects of statins. In the current study, we sought to investigate the effects of simvastatin on the Akt pathway in prostate cancer cells with respect to the regulation of various cell functions in vitro and tumor growth in vivo. Time- and dose-dependent effects of simvastatin on LNCaP (androgen-dependent) and PC3 (androgen-independent) cells indicate that treatment with simvastatin at concentrations as low as 25 μM was sufficient to inhibit serum-stimulated Akt activity. Akin to this, treatment with simvastatin significantly inhibited serum-induced cell migration, invasion, colony formation, and proliferation. Simvastatin-mediated effects on colony formation were rescued by adenovirus-mediated expression of constitutively active Akt (myristoylated Akt) in PC3 cell lines. A PC3 xenograft model performed in nude mice exhibited reduced tumor growth with simvastatin treatment associated with decreased Akt activity and reduced prostate-specific antigen (PSA) levels. Our findings demonstrate the therapeutic benefits of simvastatin for prostate cancer and suggest a link between simvastatin, regulation of Akt activity, and PSA expression in prostate tumors.

P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

Background: The significance of Pak1 in prostate cancer remains unclear. Results: Pak1 knockdown impaired prostate tumor growth via increased expression of TGFβ and reduced secretion of MMP9. Conclusions: We demonstrated that Pak1 is a more potent mediator of prostate cancer cell migration and tumor growth than Pak6, the predominant isoform in the prostate. Significance: A novel role of Pak1 in prostate cancer is identified. P21-activated kinases (Paks) are major effectors downstream of the small Rho family of GTPases. Among the six isoforms, Pak1 is the most ubiquitous and the best characterized member. Previous studies have shown that inhibition of Pak6, which is predominantly present in the prostate compared with other tissues, inhibits prostate tumor growth in vivo. Even though Pak1 has been identified in normal prostatic epithelial cells and cancer cells, its specific role in the development of prostate cancer remains unclear. We report here that highly invasive prostate cancer cells express significantly higher levels of Pak1 protein compared with non-invasive prostate cancer cells. Furthermore, prostate tumor tissues and prostate cancer metastasized to lungs showed a higher expression of Pak1 compared with normal tissues. Interestingly, Pak6 protein expression levels did not change with the invasive/metastatic potential of the cancer cells or tumors. Although inhibition of Pak1, and not Pak6, resulted in impaired PC3 cell migration, the effects of Pak1 knockdown on transendothelial migration (microinvasion), tumor growth, and tumor angiogenesis was higher compared with Pak6 knockdown. Finally, gene array data revealed reduced expression of matrix metalloproteinase 9 with the ablation of either Pak1 or Pak6 gene expression in PC3 cells, whereas protein levels of TGFβ was elevated significantly with specific modulation of Pak1 activity or ablation of the Pak1 gene. Our observations suggest that although some level of functional redundancy exists between Pak1 and Pak6 in prostate cancer cells, targeting Pak1 is a potential option for the management of prostate tumor growth, microinvasion, and metastasis.

Simultaneous modulation of the intrinsic and extrinsic pathways by simvastatin in mediating prostate cancer cell apoptosis

BackgroundRecent studies suggest the potential benefits of statins as anti-cancer agents. Mechanisms by which statins induce apoptosis in cancer cells are not clear. We previously showed that simvastatin inhibit prostate cancer cell functions and tumor growth. Molecular mechanisms by which simvastatin induce apoptosis in prostate cancer cells is not completely understood.MethodsEffect of simvastatin on PC3 cell apoptosis was compared with docetaxel using apoptosis, TUNEL and trypan blue viability assays. Protein expression of major candidates of the intrinsic pathway downstream of simvastatin-mediated Akt inactivation was analyzed. Gene arrays and western analysis of PC3 cells and tumor lysates were performed to identify the candidate genes mediating extrinsic apoptosis pathway by simvastatin.ResultsData indicated that simvastatin inhibited intrinsic cell survival pathway in PC3 cells by enhancing phosphorylation of Bad, reducing the protein expression of Bcl-2, Bcl-xL and cleaved caspases 9/3. Over-expression of PC3 cells with Bcl-2 or DN-caspase 9 did not rescue the simvastatin-induced apoptosis. Simvastatin treatment resulted in increased mRNA and protein expression of molecules such as TNF, Fas-L, Traf1 and cleaved caspase 8, major mediators of intrinsic apoptosis pathway and reduced protein levels of pro-survival genes Lhx4 and Nme5.ConclusionsOur study provides the first report that simvastatin simultaneously modulates intrinsic and extrinsic pathways in the regulation of prostate cancer cell apoptosis in vitro and in vivo, and render reasonable optimism that statins could become an attractive anti-cancer agent.

Suppression of interactions between prostate tumor cell‐surface integrin and endothelial ICAM‐1 by simvastatin inhibits micrometastasis

Cancer micrometastasis relies on the ability of cancer cells to secrete angiogenic modulators, to interact with the vascular endothelium, and to overcome the resistance offered by the endothelial‐barrier. Being an essential step prior to metastasis, blockage of micrometastasis can have potential applications in cancer therapy and metastasis prevention. Due to poorly known molecular mechanisms leading to micrometastasis, developing therapeutic strategies to target prostate cancer utilizing drugs that block micrometastasis is far from reality. Here, we demonstrate the potential benefits of simvastatin in the inhibition of prostate cancer micrometastasis and reveal the novel molecular mechanisms underlying this process. First, we showed that simvastatin inhibited the ability of human PC3 prostate cancer cells for transendothelial migration in vitro. Second, our data indicated that simvastatin modulates the expression of tumor‐derived factors such as angiopoietins and VEGF‐A at the mRNA and protein levels by the PC3 cells, thus preventing endothelial‐barrier disruption. Third, simvastatin directly activated endothelial cells and enhances endothelial‐barrier resistance. Apart from this, our study revealed that simvastatin‐mediated effect on PC3 micrometastasis was mediated through inhibition of integrin αvβ3 activity and suppression of interaction between prostate cancer cell integrin αvβ3 with endothelial ICAM‐1. J. Cell. Physiol. 228: 2139–2148, 2013.

Diltiazem Enhances the Apoptotic Effects of Proteasome Inhibitors to Induce Prostate Cancer Cell Death

Diltiazem is a calcium channel blocker used to treat cardiovascular ailments. In addition, reports suggest that diltiazem induces cell death, which could make it a drug of choice for the treatment of cancer associated with hypertension. The goal of this research was to determine whether diltiazem is capable of inducing apoptosis in prostate cancer cells, either alone or in combination with the proteasome inhibitors, lactacystin and bortezomib (Velcade). Bortezomib is approved for the treatment of multiple myeloma; unfortunately, it has side effects that limit its utility. Presumably these side effects could be decreased by reducing its dose in combination with another drug. We have previously shown that lactacystin induces apoptosis in LNCaP cells; here, we show that this effect was enhanced by diltiazem. Furthermore, in proteasome inhibitor-resistant DU145 cells, diltiazem alone did not induce apoptosis but decreased cytosolic calcium levels and induced mitochondrial fission; likewise, lactacystin did not induce apoptosis but up-regulated the proapoptotic protein Bik. However, increasing concentrations of diltiazem in combination with lactacystin or bortezomib induced apoptosis in a dose-dependent and synergistic manner. The combination of diltiazem and lactacystin also up-regulated the levels of Bik and released Bak from Bcl-xL, indicating the involvement of the Bcl2 family pathway in this apoptosis. In addition, the drug combination up-regulated GRP78, suggesting also the involvement of endoplasmic reticulum stress in the apoptotic response. Thus, our results demonstrate a potential therapeutic advantage of combining a frequently used calcium channel blocker with proteasome inhibitors in the treatment of prostate cancer.

Abstract B47: Pleiotropic effects of simvastatin on prostate tumor growth and metastasis

Background: Statins are a class of drugs that inhibits HMG-CoA reductase, a rate liming enzyme in cholesterol synthesis. Simvastatin, a widely used generic drug for preventing cardiovascular events inhibit inflammation and stabilize atherosclerotic plaques. Growing body of evidences suggest that statins have the potential to reduce the risk of many cancer types. Objectives and Hypothesis: Our long-term goal is to enable the development of new and innovative therapeutics for prostate cancer through better understanding of the molecular mechanisms regulating prostate cancer growth and bone metastasis. In prostate cancer cells, simvastatin is known to induce apoptosis. Akt, a multitask signaling molecule, is the major survival kinase activated in cancer cells. Our central hypothesis is that treatment with simvastatin will inhibit Akt affecting prostate cancer cell function, tumor growth and metastasis. The rationale for the proposed research is that, once it is known mechanistically how simvastatin regulates prostate cancer cell function, it is likely that prostate tumor growth and metastasis can be downregulated therapeutically utilizing simvastatin using a novel drug-repurposing strategy. This would be of singular importance in the management of this disease. Experimental Design and Results: In the current study, we sought to investigate the pleiotropic effects of simvastatin on major signaling pathways in prostate cancer cells with respect to the regulation of cellular functions such as migration, proliferation, colony/foci formation and invasion, along with its already known effects on apoptosis. Time- and dose-effects of simvastatin on LNCaP (androgen-dependent) and PC-3 (androgen-independent) cells indicated that treatment with as low as 25µM simvastatin was sufficient to inhibit serum-stimulated activation of Akt-mTOR and cRaf-ERK pathways. Akin to this, treatment with 25µM simvastatin significantly inhibited serum- and EGF-induced cell migration, invasion, colony formation and proliferation. Simvastatin-mediated effects on cell migration and colony formation was rescued by adenovirus-mediated expression of constitutively active Akt (myristoylated Akt) in androgen-independent prostate cancer cells lines such as PC3 and LnCAP C4-2. A xenograft model performed in nude mice exhibited reduced PC3 prostate tumor growth with simvastatin treatment (2mg/kg body weight/day for 2 weeks) demonstrating the therapeutic potential of simvastatin for prostate cancer therapy. Conclusions and Future Directions: Our findings suggest a link between simvastatin and Akt/ERK signaling in the regulation of prostate cancer growth and metastasis. Further investigation is currently underway in our laboratory to unravel the molecular mechanisms on simvastatin-mediated effects on prostate cancer leading to tumor growth and bone metastasis in vivo using transgenic mouse models such as AKt+ and TRAMP+ mice. We also plan to undertake a clinical study on patients with prostate cancer who were on statin treatment prior to and after diagnosis and analyze biopsy specimen from these patients. A prospective study will look at the role statins in prostate cancer prevention and/or on management. Our ultimate aim is to investigate if statins can be used as an adjuvant drug in the treatment of patients already diagnosed with prostate cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B47.

Suppression of interactions between prostate tumor cell integrin αvβ3 and endothelial ICAM-1 by simvastatin inhibits prostate cancer micrometastasis

Cancer micrometastasis relies on the ability of cancer cells to secrete angiogenic modulators, to interact with the vascular endothelium, and to overcome the resistance offered by the endothelial‐barrier. Being an essential step prior to metastasis, blockage of micrometastasis can have potential applications in cancer therapy and metastasis prevention. Due to poorly known molecular mechanisms leading to micrometastasis, developing therapeutic strategies to target prostate cancer utilizing drugs that block micrometastasis is far from reality. Here, we demonstrate the potential benefits of simvastatin in the inhibition of prostate cancer micrometastasis and reveal the novel molecular mechanisms underlying this process. First, we showed that simvastatin inhibited the ability of human PC3 prostate cancer cells for transendothelial migration in vitro. Second, our data indicated that simvastatin modulates the expression of tumor‐derived factors such as angiopoietins and VEGF‐A at the mRNA and protein levels by the PC3 cells, thus preventing endothelial‐barrier disruption. Third, simvastatin directly activated endothelial cells and enhances endothelial‐barrier resistance. Apart from this, our study revealed that simvastatin‐mediated effect on PC3 micrometastasis was mediated through inhibition of integrin αvβ3 activity and suppression of interaction between prostate cancer cell integrin αvβ3 with endothelial ICAM‐1. J. Cell. Physiol. 228: 2139–2148, 2013.

Suppression of interactions between prostate tumor cell-surface integrin and endothelial ICAM-1 by simvastatin inhibits micrometastasis: SIMVASTATIN AND PROSTATE CANCER MICROMETASTASIS

Cancer micrometastasis relies on the ability of cancer cells to secrete angiogenic modulators, to interact with the vascular endothelium, and to overcome the resistance offered by the endothelial‐barrier. Being an essential step prior to metastasis, blockage of micrometastasis can have potential applications in cancer therapy and metastasis prevention. Due to poorly known molecular mechanisms leading to micrometastasis, developing therapeutic strategies to target prostate cancer utilizing drugs that block micrometastasis is far from reality. Here, we demonstrate the potential benefits of simvastatin in the inhibition of prostate cancer micrometastasis and reveal the novel molecular mechanisms underlying this process. First, we showed that simvastatin inhibited the ability of human PC3 prostate cancer cells for transendothelial migration in vitro. Second, our data indicated that simvastatin modulates the expression of tumor‐derived factors such as angiopoietins and VEGF‐A at the mRNA and protein levels by the PC3 cells, thus preventing endothelial‐barrier disruption. Third, simvastatin directly activated endothelial cells and enhances endothelial‐barrier resistance. Apart from this, our study revealed that simvastatin‐mediated effect on PC3 micrometastasis was mediated through inhibition of integrin αvβ3 activity and suppression of interaction between prostate cancer cell integrin αvβ3 with endothelial ICAM‐1. J. Cell. Physiol. 228: 2139–2148, 2013.