In a recent article in Molecules, researchers from Greece investigated using multiwalled carbon nanotubes (CNTs) as carriers for PTEN (phosphatase and tensin homolog deleted on chromosome 10) and its truncated fragments in breast cancer cells.
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PTEN is a tumor suppressor protein with potential therapeutic applications in cancer treatment. The research aims to explore the efficacy of CNTs as a delivery system for PTEN variants and assess their impact on inhibiting cancer growth and inducing apoptosis.
Background
PTEN loss is common in various cancers, leading to dysregulated cell growth and survival. Understanding PTEN function and exploring novel delivery systems for PTEN-based therapies is crucial for developing effective cancer treatments. Previous studies have highlighted the importance of the C2 domain in PTEN's C-terminal region for its tumor-suppressive activities.
The Current Study
The full-length human PTEN (hPTEN1) and its truncated forms, hPTEN2 and hPTEN3, were cloned and overexpressed in Escherichia coli (E. coli) cells. The sequences encoding these PTEN variants were inserted into expression vectors for protein production. The recombinant proteins were then purified using affinity chromatography under denatured conditions to ensure high purity and yield.
To facilitate the delivery of PTEN variants into breast cancer cells, the purified proteins were immobilized on the surface of multiwalled CNTs. The CNTs were chosen for their large surface area, small size, and ability to penetrate lipid membranes effectively. Before protein immobilization, the CNTs were surface decorated with polyethylene glycol (PEG) to enhance biocompatibility and hydrophilicity.
Breast cancer cell lines, including T-47D, MCF-7, and PTEN-deficient ZR-75-1 cells, were cultured in appropriate media and conditions. The cells were treated with CNTs functionalized with full-length PTEN (hPTEN1) or truncated forms (hPTEN2 and hPTEN3) at varying concentrations. Cell viability assays and molecular analyses were performed to assess the effects of PTEN variants on cell proliferation and apoptosis.
Cell viability was assessed using standard assays such as MTT or MTS to measure metabolic activity and cell proliferation. Apoptosis induction was evaluated through Annexin V staining or TUNEL assays to detect apoptotic cells. The expression levels of critical apoptotic markers were analyzed using techniques like Western blotting and RT-PCR to elucidate the molecular mechanisms underlying PTEN-mediated effects on cancer cells.
Statistical analysis was conducted using appropriate software, such as GraphPad Prism, to determine the significance of the results. Data were presented as mean ± standard deviation, and statistical significance was defined based on p-values (p < 0.01 represented by **, p < 0.001 represented by ***). Each experiment was performed in triplicate or as specified to ensure reproducibility and reliability of the findings.
Results and Discussion
The study revealed that the full-length PTEN variant (hPTEN1) immobilized on multiwalled CNTs significantly inhibited cancer cell growth in breast cancer cell lines, particularly ZR-75-1 and MCF-7 cells.
This observation underscores the potent tumor-suppressive activity of full-length PTEN when delivered using CNTs as carriers. In contrast, truncated PTEN fragments, such as hPTEN2 and hPTEN3, exhibited a lesser effect on inhibiting cell proliferation. These findings suggest that the structural integrity of full-length PTEN, including the interaction with the C2 domain in the C-terminal tail, is crucial for its anti-cancer efficacy.
Treatment with full-length PTEN-CNTs inhibited cancer cell growth and stimulated apoptosis in breast cancer cells. The alteration of expression levels of key apoptotic markers further supported the pro-apoptotic effects of PTEN in cancer cells. The study highlighted the importance of PTEN in modulating apoptotic pathways and regulating cell survival, emphasizing its potential as a therapeutic target in cancer treatment.
The comparison between full-length PTEN and its truncated fragments provided valuable insights into the nuanced functionality of PTEN in cancer cells. While truncated PTEN fragments retained the active site, their reduced ability to interact with cellular membranes or potential protein degradation may have contributed to their diminished anti-cancer effects. The presence of critical domains, such as the CBR3 loop and the C2 domain, in full-length PTEN appears essential for its optimal tumor-suppressive activity.
Conclusion
The research underscores the potential of CNTs as effective carriers for delivering PTEN variants in cancer therapy. The study provides valuable insights into the mechanisms of PTEN-mediated tumor suppression and the role of CNTs in enhancing therapeutic outcomes. These findings contribute to the development of advanced strategies for delivering tumor suppressor proteins in cancer treatment, emphasizing the need for further research in this promising field.
Journal Reference
Papi, RM., et al. (2024). Carbon Nanotube-Mediated Delivery of PTEN Variants: In Vitro Antitumor Activity in Breast Cancer Cells. Molecules. doi.org/10.3390/molecules29122785