Activation of the Complement System for Cancer Treatment through Telomerase Overexpression; By Ignacio Julián García - Muñoz Yáñez Abstract Telomerase is an enzyme that is present in male sperm cells to ensure maintenance of telomere length at maximum levels during spermatogenesis, but it’s also present overexpressed in the majority of cancer cells, allowing them to evade cellular aging and contribute to immortality. I propose the strategy of targeting the cancer cells by utilizing the activation of the complement system based on this telomerase overexpression. Introduction Telomerase is the enzyme responsible for attaching repetitive nucleotide sequences (TTAGG), to the ends of the chromosomes, which are known as telomeres. On the main, somatic cells have no telomerase or only low amounts which makes the telomeres to be shortened from each cell division stage and at last cause the genetic damage and cellular apoptosis. Nevertheless, the majority of tumor cells have marked telomerase overexpression which enables them to keep their telomeres intact and escape the natural aging process. This mechanism basically illustrates the so-called “cellular immortality ” whereby cancer cells are able to multiply without any limitation. Hence, telomerase has been a focal point of great interest in anti-cancer therapy aimed at its inhibition as a means of aiding the immune system in the destruction of tumor cells while avoiding injury to normal cells. Justification of the Proposal Cancer Cell Marker: Telomerase is an important enzyme, and its main function is the maintenance of telomere length which is crucial for the non-destructive replication of essential DNA during cellular process. In the largest percentage of normal cells, telomerase is turned off which leads to the continuously shortening of telomeres. According to the information obtained from recent studies, 80-90% of human tumors develop telomerase overexpression, which gives cancer cells an incredible ability to replicate infinitely and thus escape senescence. Such nature of telomerase makes it an additional diagnostic marker for the discovery of oncogenic cells along with the help of a targeted treatment involving the destruction of cancer cells only. The immune system activation of the complement system involves a sequence of proteins that work together in the opsonization of pathogens and the stimulation of inflammation. Impressionable cancer cells can be removed from the body by a process called opsonization, in which to the complement system if activated, they can be fitted with a tag for immune cells thus making them more likely than the others to get destroyed. Also, the organization of the membrane attack complex (MAC) can lead to the direct killing of the tumor cells. Thus, the combination of telomerase-targeting therapies and complement activation may selectively destroy the cancer cells presenting increased telomerase levels. Nevertheless, a major difficulty in cancer care is the unwished implications of the healthy cell damages through the application of treatments that are noticeable and will produce side effects as well. When we study merely the overactivity of telomerase as a biomarker for tumor cells, we minimize the consequences on normal tissues. Considering that telomerase is mainly sluggish in healthy somatic cells, the types of therapies targeting this enzyme can selectively reach cancer cells but not other tissues, thus, toxicity from such therapies may be significantly reduced. Also, such an approach may result in higher efficiency and lower toxicity as compared to the traditional chemotherapies, which are often aimed at destroying highly proliferating cells of the body without any specificity. Proposed Methodology Telomerase Detection: Testing is the most reliable and common procedure of quantifying telomerase activity in the cell, and a method that is often used for the goal is the Telomeric Repeat Amplification Protocol (TRAP) assay Steps Involved: 1. Cell Extraction: Obtaining the cell lysates from cancer tissues or cultured-lab-grown cancer cells. 2. PCR Amplification: Use a specific primer designed to amplify the telomeric repeats. These ones typically consist of a primer that anneals to the telomeric sequence and a reverse primer that binds with a neighbor region. 3. Gel Electrophoresis: Separate the PCR products on an agarose gel to visualize and measure the telomerase activity. A ladder of bands corresponding to different telomere lengths indicates the presence and activity level of telomerase Exact Markers: The hTERT subunit of telomerase is the most important marker for detection. Specific primers targeting the hTERT mRNA can also be used in quantitative RT-PCR (qRT-PCR) assays to size up the expression level of telomerase in cancer cells. Complement Activation: To activate the complement system in response to telomerase overexpression, a potential approach is to design synthetic peptides or antibodies that specifically target the hTERT protein or its associated structures. Outline of Method: 1. Antibody Development: Creating monoclonal antibodies against hTERT that are able to bind only with cancer cells overexpressing telomerase. (e.g., by including complement-binding sites). 2. Complement Activation: Once the antibodies bind to the telomerase on the surface of cancer cells, the classical pathway of complement activation starts, eventually resulting in the opsonization of these cancer cells, marking them for destruction by the phagocytic cells. 3. Modification of Complement Proteins: Alternatively, arrange complement proteins (e.g., C3b) to enhance their binding hTERT, allowing the specific activation of the complement cascade upon detection of this telomerase overexpression. Validation in Preclinical Models To evaluate the efficacy of this proposed combinated therapy, preclinical studies can be considered, using both in vitro cell cultures and in vivo animal models. In Vitro Studies: Use cancer cell lines with known telomerase activity (e.g., HeLa, A549) to test the combination of telomerase-targeting agents and complement activation. Assess cell viability, apoptosis rates, and telomere length changes during and after the treatment. Conduct experiments with immune cells (e.g., macrophages) to evaluate how effectively the opsonized cancer cells are phagocytosed compared to untreated cells. In Vivo Studies: Employ xenograft mouse models, where human cancer cells expressing high levels of telomerase are implanted. Treat these mice with the designed antibodies or synthetic peptides combined with a complement activation strategy. Monitor tumor growth, overall survival, and any adverse effects on normal tissues to assess the specificity and effectiveness of the treatment. Challenges and Considerations Specificity: Ensuring the specificity of the proposed therapy is crucial to avoid targeting normal cells, particularly reproductive cells and zygotes, which also express telomerase at certain stages. This challenge arises because some normal somatic cells can exhibit low levels of telomerase activity, especially in stem cells and germline cells involved in reproduction. To enhance specificity: ● Targeting Strategies: Use of monoclonal antibodies or synthetic peptides that are highly specific for the hTERT, predominantly expressed in cancer cells. This could help minimize the off-target effects ● Conditional Expression Systems: Employ genetic engineering techniques to create a conditionally active complement system that only activates in the presence of cancer-specific markers, thereby avoiding normal cells and reproductive cells. ● Biomarker Profiling: Combine telomerase detection with additional biomarkers unique to cancer cells to refine targeting. For instance, using markers like CD133 or EpCAM that are often overexpressed in cancer stem cells could provide an additional layer of specificity. Tumor Resistance Cancer cells may develop mechanisms to evade the immune response and treatment strategies, including: ● Downregulation of Antigen Expression: Some kinds of tumors may not have that overexpression of telomerase, are able to reduce the expression of it or related antigens to escape detection. ● Complement Inhibition: Tumor cells can produce proteins that inhibit complement activation or recruit regulatory immune cells that start the immune response. To address these issues: ● Combination Therapies: Incorporate other therapeutic agents alongside the telomerase-targeted complement activation to enhance this treatment efficacy. (e.g., immune checkpoint inhibitors) ● Monitoring Resistance Mechanisms: Regularly monitor tumors for changes in the amount of telomerase expression and other biomarkers to adapt the treatment if necessary. Safety While targeting telomerase may offer a more selective approach, potential side effects must still be assessed. Risks may include: ● Off-Target Effects: If the therapy accidentally affects normal cells with low telomerase activity, it could lead to unwanted damage. ● Immune Reactions: Activation of the complement system could result in unwanted inflammatory responses or tissue injury, particularly if healthy tissues are inadvertently targeted as cancerous. To mitigate safety concerns: ● Preclinical Testing: Make studies in cell culture and animal models to evaluate the therapeutic efficiency and identify any new adverse effects. ● Dosage Optimization: Adjust the dosage to maximize efficacy while minimizing toxicity. This may involve adapting it based on patient response to the treatment. ● Patient Monitoring: Implement rigorous monitoring protocols for patients undergoing treatment to quickly identify and manage any adverse effects or complications. Conclusions: Utilizing telomerase, as an activation of the complement system presents a different way of cancer therapy. This novel idea is grounded in the overexpression of telomerase in cancer cells and its effect on treatment, from specific and efficiency to no harm to the healthy cells. In addition to the reliability of cancer diagnosis, telomerase also plays a role in the immortality of cancer cells, a reason for which it is an extremely attractive target for therapeutic interventions. Introduction of the complement system to this strategy could employ the natural immune mechanisms of the body to distinguish and then terminate the cancer cells. In this way, the fight would consist of a double action which may make it more effective in the treatment of tumors that are resistant to traditional therapies. My ideas, such as those showing the role of the immune system in activating tumor cells, would be the footsteps to the much-needed cancer management improvements. Abbreviations: TTAGGG : DNA sequence repeated thousands of times that the telomeres have at the ends of chromosomes MAC (Membrane Attack Complex) TRAP (Telomeric Repeat Amplification Protocol) PCR (Polymerase Chain Reaction) hTERT (human Telomerase Reverse Transcriptase) mRNA (Messenger RNA) qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction) C3b: Protein fragment generated during the activation of the complement system HeLa : Human cell line derived from cervical cancer cells taken from Henrietta Lacks in 1951. A549 : Human lung carcinoma cell line used in cancer research. CD133 : Glycoprotein and a marker for cancer stem cells. EpCAM (Epithelial Cell Adhesion Molecule): Cell surface protein involved in cell-cell adhesion. Often overexpressed in various types of cancer. References: BioMed Central, MDPI World Health Organization (WHO). Cancer. National Cancer Institute (NCI). Telomerase. Shay, J. W., & Wright, W. E. (2019). " Telomeres and Telomerase: A Review. 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