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Oncology

Epigenetic Processes in Cancer Research

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Epigenetic Processes in Cancer Research

Abstract

Epigenetics is the study of the phenotypic variation caused by external factors (e.g. diet, nicotine use, and carcinogenic chemical exposure) that influence the mechanisms through which cells read and interpret genes. Epigenetic modifications are independent of genetic mutations. Currently, epigenetics offers significant promise in novel noninvasive cancer therapies and early diagnostic tools. Key epigenetic processes including DNA methylation and histone modification are reversible, unlike most genetic mutations. Reversing these processes in tumor suppressor genes can restore normal behavior in tumor cells. This review discusses the biological basis and treatment potential of these processes and provides a brief analysis of their potential application in cancer treatment.

Introduction

DNA is a double-helical structure packed into the nuclei of eukaryotic cells in the form of chromatin. The basic unit of DNA packing in chromatin is the nucleosome, a complex of eight histone proteins and approximately 140 DNA base pairs. Further coiling of repeating nucleosome fibers eventually yields a chromatid, a key component of a chromosome.

Gene expression is epigenetically regulated through alterations in chromatin structure and the double helix of DNA by addition of functional groups. DNA methylation and histone modification are the most understood mechanisms of such epigenetic modulation. Errant modification of functional group patterns in DNA or histone tails could result in unintended silencing of critical regulatory genes, a process which contributes to the development of several diseases, including cancer. Understanding these complex mechanisms is essential for the development of cancer therapies that reverse the inhibition of tumor-suppressor genes. This review seeks to outline the biochemical basis of the gene silencing effected by DNA methylation and histone modification, as well as the current developments in reversing these mechanisms for the prevention of cancer.

Histone Modification & Its Implications on Cancer

Histone modifications are reversible modifications at the N-terminals of histones in a nucleosome. These include, but are not limited to, acetylation, methylation, phosphorylation, and ubiquitination. Depending on the group added or removed from a given histone, local gene transcription can be upregulated or downregulated. Histone acetylation, for instance, modifies chromatin into a less-condensed conformation that is more transcriptionally active. Conversely, histone deacetylases (HDACs) remove acetyl groups, increasing the ionic attractions between positively charged histones and negatively charged DNA and tightening chromatin structure. In order to further condense chromatin, HDACs can also recruit K9 histone methyltransferases (HMTs) to methylate H3K9 histone residues, providing the condensing agent heterochromatin protein 1 (HP1) with a binding site. As a result of the condensed heterochromatin conformation, the cell has limited transcriptional access to the genome.

Recent research has established histone modifications as useful biomarkers in cancer diagnosis. HDACs, specifically HDAC1, can often be identified in elevated quantities in prostate and gastric cancer patients. Prostate cancer tumors have been characterized by general hypomethylation of histone residues H4K20me1 and H4K20me2, as well as hypermethylation of residue H3K27 and increased activity of its specific methyltransferase. These conditions are now associated with progression and metastasis of prostate cancer. Histone modifications play a multifaceted role in cancer diagnosis and treatment. In addition to serving as diagnostic biomarkers, they are also potential therapeutic agents. Drugs that specifically inhibit HDACs are currently being manufactured and assessed for clinical application after recent FDA approval.5

DNA Methylation & Its Implications on Cancer

In DNA methylation, a methyl group is covalently added to a cytosine ring in the DNA sequence, forming a 5-methylcytosine. Molecules of 5-methylcytosine are found primarily at cytosine-guanine dinucleotides (CpGs). Some areas of high CpG density, such as CpG islands, are characteristically unmethylated and are located in or near the promoter regions of genes, allowing them to play a role in gene expression. Enzymes termed DNA methyltransferases (DNMTs) facilitate the methylation of CpG residues. Three key members of this family include Dnmt1, Dnmt3a, and Dnmt3b. Dnmt1 is labeled as the “maintenance” DNMT due to its methylation of DNA near replication forks, which preserves the epigenetic inheritance of methylation patterns across cellular generations. The other two DNMTs serve to directly methylate other CpG residues. These enzymes can either be recruited by a transcription factor bound to the promoter region of a given gene to methylate a specific CpG island or simply methylate all CpG sites across a genome not protected by a transcription factor. Several families of proteins, such as the UHRF proteins, the zinc-finger proteins, and most notably, the MBD proteins, are responsible for the interpretation of these methylation patterns. For example, MBD proteins possess transcriptional repression domains that allow them to bind to methylated DNA and silence nearby genes.

For a malignant cancer to develop, critical tumor-suppressor genes such as p53 must be knocked out. This process can be accomplished through hypermethylation of CpG islands in certain promoter regions and genes. For example, when the DNA repair gene BRCA1 becomes hypermethylated, it is rendered inactive, which can lead to the development of breast cancer. The reversibility of methylation implies that these tumor-suppressor genes could be reactivated to effectively treat tumors. Infiltration of a cell by pharmaceutical agents that inhibit methylation-mediated suppression could restore the function of a silenced tumor-suppressor gene.6

One recent study demonstrated that azacitidine, an agent that can be incorporated into DNA, is capable of inducing hypomethylation. The chemical modification of this agent’s diphosphate form by a ribonucleotide reductase and subsequent phosphorylation creates a triphosphate form that displaces cytosine bases in DNA. As a result, DNMTs are limited in methylation functionality since they are isolated on a substituted DNA strand.7 Such manipulation of naturally occurring DNA methylation processes in cancer cells appears to be a promising method of restoring the normal function of tumor-suppressor genes.

Co-Application of DNA Methylation & Histone Modification

The discovery of the protein MeCP2 has validated the hypothesis that DNA methylation regulates histone acetylation patterns. MeCP2 recruits histone HDACs to the promoter regions of methylated CpG islands. The resulting hypoacetylated histones yield a highly condensed heterochromatin structure that represses transcription of nearby genes. Clinical studies have thus emphasized the utilization of both demethylating agents and histone deacetylase inhibitors for transcriptional activation of tumor-suppressor genes.8

Conclusion

The field of epigenetics has opened a door to novel, promising cancer therapies unimaginable just a decade ago. Manipulating DNA methylation and histone modification can reverse tumor-suppressor gene silencing. Reactivating tumor-suppressor genes through epigenetic modifications can halt and reverse cancer proliferation.

References

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Developments in Gold Nanoparticles and Cancer Therapy

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Developments in Gold Nanoparticles and Cancer Therapy

Abstract

Nanotechnology has recently produced several breakthroughs in localized cancer therapy. Specifically, directing the accumulation of gold nanoparticles (GNPs) in cancerous tissue enables the targeted release of cytotoxic drugs and enhances the efficacy of established cancer therapy methods. This article will give a basic overview of the structure and design of GNPs, the role of GNPs in drug delivery and localized cancer therapy, and the challenges in developing and using GNPs for cancer treatment.

Introduction

Chemotherapy is currently the most broadly utilized method of treatment for most subtypes of cancer. However, cytotoxic chemotherapy drugs are limited by their lack of specificity; chemotherapeutic agents target all of the body’s most actively dividing cells, giving rise to a number of dangerous side effects.1 GNPs have recently attracted interest due to their ability to act as localized cancer treatments—they offer a non-cytotoxic, versatile, specific targeting mechanism for cancer treatment and a high binding affinity for a wide variety of organic molecules.2 Researchers have demonstrated the ability to chemically modify the surfaces of GNPs to induce binding to specific pharmaceutical agents, biomacromolecules, and malignant cell tissues. This allows GNPs to deliver therapeutic agents at tumor sites more precisely than standard intravenous chemotherapy can. GNPs also increase the efficiency of established cancer therapy methods, such as hyperthermia.3 This article will briefly cover the design and characteristics of GNPs, and then outline both the roles of GNPs in cancer therapy and the challenges in implementing GNP-based treatment options.

Design and Characteristics of Gold Nanoparticles

Nanoparticle Structure

To date, gold nanoparticles have been developed in several shapes and sizes.4 Although GNPs have also successfully been synthesized as rods, triangles, and hexagons, spherical GNPs have been demonstrated to be one of the most biocompatible nanoparticle models. GNP shape affects accumulation behavior in cells.4 A study by Tian et. al. found that hexagonal GNPs produced a greater rate of vesicular aggregation than both spherical and triangular GNPs.

Differences in GNP shape also cause variation in surface area and volume, which affects cellular uptake, biocompatibility, and therapeutic efficiency.4 For example, GNPs with greater surface area or more vertices possess enhanced cell binding capabilities but also heightened cell toxicity. Clearly, GNP nanoparticles must be designed with respect to their intended function.

Nanoparticle Surface Modification

In order to target specific cells or tissues, GNPs must undergo a ligand attachment process known as surface modification. The types of ligand particles attached to a GNP affect its overall behavior. For example, ligand particles consisting of inert molecular chains can stabilize nanoparticles against inefficient aggregation.5 Polyethylene glycol (PEG) is a hydrocarbon chain that stabilizes GNPs by repelling other molecules using steric effects; incoming molecules are unable to penetrate the PEG-modified surface of the GNPs.5 Certain ligand sequences can enable a GNP to strongly bind to a target molecule by molecular recognition, which is determined by geometric matching of the surfaces of the two molecules.5

Tumor cells often express more cell surface receptors than normal cells; targeting these receptors for drug delivery increases drug accumulation and therapeutic efficacy.6 However, the receptors on the surface of tumor cells must be exclusive to cancerous cells in order to optimize nanoparticle and drug targeting. For example, most tumor cells have integrin receptors.7 To target these residues, the surfaces of the therapeutic GNPs can be functionalized with the arginine-glycine-aspartic acid (RGD) sequence, which binds to key members within the integrin family.8 Successful targeting can lead to endocytosis and intracellular release of the therapeutic elements that the GNPs carry.

An important factor of GNP therapy is the efficient targeting and release of remedial agents at the designated cancerous site. There are two types of GNP targeting: passive and active. In passive targeting, nanoparticles accumulate at a specific site by physicochemical factors (e.g. size, molecular weight, and shape), extravasation, or pharmacological factors. Release can be triggered by internal factors such as pH changes or external stimuli such as application of light.2 In active targeting, ligand molecules attached to the surface of a GNP render it capable of effectively delivering pharmaceutical agents and large biomacromolecules to specific cells in the body.

Gold Nanoparticles in Localized Cancer Therapy

Hyperthermia

Hyperthermia is a localized cancer therapy in which cancerous tissue is exposed to high temperatures to induce cell death. Placing gold nanoparticles at the site of therapy can improve the efficiency and effectiveness of hyperthermia, leading to lower levels of tumor growth. GNPs aggregated at cancerous tissues allow intense, localized increases in temperature that better induce cell death. In one study on mice, breast tumor tissue containing aggregated GNPs experienced a temperature increase 28°C higher than control breast tumor tissue when subjected to laser excitation.9 While the control tissue had recurring cancerous growth, the introduction of GNPs significantly increased the therapeutic temperature of the tumors and permanently damaged the cancerous tissue.

Organelle Targeting

GNPs are also capable of specifically targeting malfunctioning organelles in tumor cells, such as nuclei or mitochondria. The nucleus is an important target in localized cancer therapy since it controls the processes of cell growth, proliferation, and apoptosis, which are commonly defective in tumor cells. Accumulation of GNPs inside nuclei can disrupt faulty nuclear processes and eventually induce cell apoptosis. The structure of the GNP used to target the nucleus determines the final effect. For example, small spherical and “nanoflower”-shaped GNPs compromise nuclear functioning, but large GNPs do not.10

Dysfunctional mitochondria are also valuable targets in localized therapy as they control the energy supply of tumor cells and are key regulators of their apoptotic pathways.10 Specific organelle targeting causes internal cell damage to cancerous tissue only, sparing normal tissue from the damaging effects of therapeutic agents. This makes nuclear and mitochondrial targeting a desirable treatment option that merits further investigation.

Challenges of Gold Nanoparticles in Localized Cancer Therapy

Cellular Uptake

Significant difficulties have been encountered in engineering a viable method of cellular GNP uptake. Notably, GNPs must not only bind to a given cancer cell’s surface and undergo endocytosis into the cell, but they must also evade endosomes and lysosomes.10 These obstacles are present regardless of whether the GNPs are engineered to target specific organelles or release therapeutic agents inside cancerous cells. Recent research has demonstrated that GNPs can avoid digestion by being functionalized with certain surface groups, such as polyethylenimine, that allow them to escape endosomes and lysosomes.10

Toxic Effects on Local Tissue

The cytotoxic effects of GNPs on local cells and tissues remain poorly understood.11 However, recent research developments have revealed a relationship between the shapes and sizes of GNPs and their cell toxicities. Larger GNPs have been found to be more cytotoxic than smaller ones.12 Gold nanospheres were lethal at lower concentrations, while gold nanostars were less toxic.13 While different shapes and sizes of GNPs can be beneficial in various localized cancer therapies, GNPs must be optimized on an application-by-application basis with regard to their toxicity level.

Conclusion

Gold nanoparticles have emerged as viable agents for cancer therapy. GNPs are effective in targeting malignant cells specifically, making them less toxic to normal cells than traditional cancer therapies. By modifying their surfaces with different chemical groups, scientists can engineer GNPs to accumulate at specific tumor sites. The shape and size of a GNP also affect its behavior during targeting, accumulation, and cellular endocytosis. After accumulation, GNPs may be used to enhance the efficacy of established cancer therapies such as hyperthermia. Alternatively, GNPs can deliver chemotherapy drugs to tumor cells internally or target specific organelles inside the cell, such as the nucleus and the mitochondria.

Although some research has shown that GNPs themselves do not produce acute cytotoxicity in cells, other research has indicated that nanoparticle concentration, shape, and size may all affect cytotoxicity. Therefore, nanoparticle design should be optimized to increase cancerous cell death but limit cytotoxicity in nearby normal cells.

References

  1. Estanquiero, M. et al. Colloid Surface B. 2015, 126, 631-648.
  2. Pissuwan, D. et al. J Control Release. 2009, 149, 65-71.
  3. Chatterjee, D. V. et al. Ther Deliv. 2011, 2, 1001-1014.
  4. Tian, F. et al. Nanomedicine. 2015, 10, 2643-2657.
  5. Sperling, R.A. et al. Phil Trans R Soc A. 2010, 368, 1333-1383.
  6. Amreddy, N. et al. Int J of Nanomedicine. 2015, 10, 6773-6788.
  7. Kumar, A. et al. Biotechnol Adv. 2013, 31, 593-606.
  8. Perlin, L. et al. Soft Matter. 2008, 4, 2331-2349.
  9. Jain, S. et al. Br J Radiol. 2012, 85, 101-113.
  10. [Kodiha, M. et al. Theranostics. 2015, 5, 357-370.
  11. Nel, A. et al. Science. 2006, 311, 622-627.
  12. Pan, Y. et al. Small. 2007, 3, 1941-1949.
  13. Favi, P. M. et al. J Biomed Mater Red A. 2015, 103, 3449-3462. 

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