Study 1: Do Cannabinoids alter gene expression in humans and if so, how?

Introduction to Epigenetics

Epigenetics is a field of biology that explores how gene expression can be regulated without altering the underlying DNA sequence. In other words, epigenetics studies changes in gene activity and expression that are not caused by changes in the DNA sequence itself. Instead, these changes are influenced by various factors, including environmental factors, lifestyle choices, and developmental stages.

The term "epigenetics" is derived from the Greek word "epi," meaning "above" or "on top of," indicating that these changes occur "on top of" or in addition to the DNA sequence. Epigenetic modifications can affect how genes are turned on or off, thereby influencing the production of proteins and ultimately impacting various biological processes and traits.

There are several mechanisms through which epigenetic modifications can occur. These include DNA methylation, histone modifications, and non-coding RNAs. DNA methylation involves the addition of a methyl group to specific locations on the DNA molecule, which can inhibit gene expression. Histone modifications involve chemical changes to the proteins called histones around which DNA is wrapped, altering the accessibility of the DNA and influencing gene expression. Non-coding RNAs, such as microRNAs, orphan-noncoding RNAs and long non-coding RNAs, can regulate gene expression by interacting with messenger RNAs or directly influencing chromatin structure.

Epigenetic changes play crucial roles in various biological processes, including development, cell differentiation, and disease susceptibility. They are also increasingly recognized as important contributors to the inheritance of traits across generations, a phenomenon known as transgenerational epigenetic inheritance.

Research in epigenetics has expanded our understanding of how environmental factors, such as diet, stress, and exposure to toxins, can influence gene expression and contribute to the development of diseases like cancer, diabetes, and neurological disorders. Understanding epigenetic mechanisms holds promise for the development of novel therapeutic strategies and interventions aimed at modulating gene expression to treat or prevent disease.

Cannabinoids and Epigenetics

The relationship between epigenetics and cannabinoids, specifically the more than 100 active compounds found in cannabis, is an area of growing interest in scientific research. Cannabinoids, such as tetrahydrocannabinol (THC) and cannabidiol (CBD), exert their effects by interacting with the endocannabinoid system, a complex signaling network involved in regulating various physiological processes.

Studies have shown that cannabinoids can influence epigenetic mechanisms, thereby impacting gene expression and cellular function. One of the primary ways cannabinoids exert epigenetic change is through modulation of DNA methylation and histone modifications.

For example, research has demonstrated that THC, the main psychoactive component of cannabis, can alter DNA methylation patterns in various regions of the genome. Changes in DNA methylation can affect gene expression, potentially leading to alterations in cellular function and behavior. There is little doubt that cannabinoids induce changes that are beneficial. The question is how?

Similarly, cannabinoids have been shown to influence histone modifications, which can also impact gene expression. For instance, CBD has been found to alter histone acetylation levels, which can regulate the accessibility of DNA and affect gene transcription.

The interaction between cannabinoids and epigenetic mechanisms is particularly relevant in the context of neurological disorders, such as epilepsy, Alzheimer's disease, and schizophrenia, where cannabinoids have shown therapeutic potential. By modulating epigenetic processes, cannabinoids may exert their therapeutic effects by restoring normal gene expression patterns and cellular function.

Furthermore, there is growing interest in the role of epigenetics in the long-term effects of cannabis use, particularly concerning cognitive function and mental health. Chronic exposure to cannabinoids may lead to persistent or permanent changes in epigenetic marks.

Overall, research into the interplay between cannabinoids and epigenetics is still in its early stages, and further studies are needed to fully understand the molecular mechanisms underlying these interactions. However, this area of research holds promise for elucidating the therapeutic potential of cannabinoids and developing novel treatments for various medical conditions.

CBXGEN was founded to help fund this type of research!

Background

Through our partnership with TopoGEN, Inc. and from the direct purchase of our products, we are able to utilize and study a highly specialized type of cancer cells, called HeLa DIM cells, which give us direct visualization and quantification of epigenetic modifications to the cells being treated with a particular drug or compound. This is achieved through observing expression of a 'reporter' gene called GFP or green fluorescence protein in the HeLa DIM cells. Reporter genes allow us to monitor the activity of these epigenetic alterations in real-time by observing the increase or decrease in the GFP intensity based on the amount of the GFP protein being expressed in each cell. When the DNA is methylated it will show a decrease in GFP expression; conversely when the DNA is demethylated there will be an increase in GFP expression, which we can measure through highly specialized equipment. GFP is a rapid and effective monitor readout for DNA methylation status of the reporter gene.

HeLa DIM Cell FACS sorting plot showing two distinct groups expressing GFP DIM cells (left) and Bright Cells(right)

EVOS image showing HeLa DIM Cells that reveal different GFP expression patterns (DIM vs. Bright)

Flow Cytometry (FACS) counts and measures GFP in cells one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. We are looking specifically at the GFP florescence activity after a colony of cultured cells has been treated with a predetermined amount of a given cannabinoid. Using a high powered EVOS fluorescence microscope, we can image live cells in real time to get an immediate readout of the cells expressing the GFP protein and their level of expression. Since we are viewing living cells, we also get a good look at the overall health and growth of the cells before and after being treated with a specific cannabinoid. The imaging (lower) and Flow readout (top) clearly show the differences between Dim and Bright cells.

Experiment A.

In this analysis, we asked a simple question: Do any of the known, purified cannabinoids alter the epigenetics of a human cell? Our system is designed to rigorously answer this question by analyzing the GFP expression profiles of Dim and Bright cells as described above. The graphs below are measuring populations of Dim and Bright cells in the population of cells before or after cannabinoid treatment. The control sample of untreated cells is shown in Panel A. Roughly half of the cells are 'Bright' or unmethylated cells while the remaining are "Dim" or methylated GFP. This experiment tests whether different cannabinoids CB(D,G,N,C) have the ability to shift or change the Dim: Bright ratio. For example, if the Dim cells are shifted to Bright cells, we would logically conclude that methylation of reporter gene (GFP) is being reduced. In contrast, if the Bright cell population is suppressed, we would conclude that the cannabinoid is stimulating methylation of the reporter.

A negative control shows about 38% cells are Bright and 62% are Dim cells (ie, roughly half and half).

A negative control shows about 38% cells are Bright and 62% are Dim cells (ie, roughly half and half).

CBG and CBN are similar to CBD but are not as potent in stimulating DNA methylation.

CBC was quite different in response. The Bright population was clearly elevated, showing that CBC is stimulating the removal of methyl groups from DNA.

This experiment is important because it shows: 1). Cannabinoids can and do alter epigenetic profiles; 2) Epigenetic revisions take place in the context of living cells and require several days to act; 3) The effect on epigenetics is different with each cannabinoid; 4) These studies open up a new field of study to find which genes might be altered by cannabinoids in normal, healthy tissues compared to diseased cells (cancer, for example); 5) All cells were healthy and dividing normally throughout the 5-day experiment (not shown). In other words, the cells were fully viable over the course of the experiment, which is important to demonstrate.

Study 1. Key Findings

  • CBXGEN has developed interrogation tools for the epigenome and specifically for following DNA methylation revision in live cells.

  • We show that cannabinoids have the ability to modify DNA methylation patterns in a model gene (GFP) suggesting that they very likely can alter expression of genes potentially occurring within different tissues of the human body.

  • Study 1 establishes a new area of cannabinoid pharmacology: the study of epigenetic gene expression modifications and how these changes could be used to treat different human diseases or alter specific pathologic disease processes.

  • For the first time, we show that different cannabinoids each have a differential impact on epigenetics, opening up new inroads for drug development originating from hemp.

  • Please continue to purchase our products and help support this vital research, thank you!