Strategy For Cancer Gene Therapy Research Articles

Emerging Role of Gene Therapy in Cancer

Cancer is one of the leading causes of death in the world, and researchers have been persistently studying the treatment of cancer. Although traditional treatment methods such as surgery, chemotherapy and radiotherapy are effective, they are often accompanied by serious side effects, drug resistance or limited effects on certain types of cancer. Therefore, it is urgent to develop more accurate and effective treatment plans. Cancer gene therapy is an innovative method to treat or prevent cancer by introducing or editing genetic material. Gene therapy brings new hope to the treatment of cancer, repairing mutant genes, activating the immune system or directly inducing cancer cell death. This review introduces some hot strategies of cancer gene therapy, including suicide gene therapy, tumor suppressor gene activation, immunotherapy, inhibition of oncogene activation, antiangiogenic gene therapy. The principles, application examples and advantages and disadvantages of these therapies in clinical research are also discussed. In addition, the challenges faced by gene therapy in improving safety and reducing side effects, and points out the direction of future research are also described, aiming to promote the transformation of these innovative therapies into conventional clinical applications.

Utilizing Adenovirus Knob Proteins as Carriers in Cancer Gene Therapy Amidst the Presence of Anti-Knob Antibodies.

Numerous gene therapy drugs for cancer have received global approval, yet their efficacy against solid tumors remains inadequate. Our previous research indicated that the fiber protein, a component of the adenovirus capsid, can propagate from infected cells to neighboring cells that express the adenovirus receptor. We hypothesize that merging this fiber protein with an anti-cancer protein could enable the anti-cancer protein to disseminate around the transfected cells, presenting a novel approach to cancer gene therapy. In our study, we discovered that the knob region of the adenovirus type 5 fiber protein is the smallest unit capable of spreading to adjacent cells in a receptor-specific manner. We also showed that the recombinant knob protein infiltrates cells after dispersing to surrounding cells. To assess the potential of the knob protein to augment gene therapy for solid tumors in mice, we expressed a fusion gene of the A subunit of cytotoxic cholera toxin and the knob region in mouse tumors. We found that this fusion protein only inhibited tumor growth in receptor-expressing mouse melanomas, and this inhibitory effect persisted even in mice with anti-knob antibodies. Our study's findings propose a novel cancer gene therapy strategy that enhances therapeutic effects by specifically delivering therapeutic proteins, expressed from in vivo administered genes, to target molecules. This outcome offers a fresh perspective on gene therapy for solid cancers, and we anticipate that knob proteins will serve as a platform for this method.

NF-κB-activated oncogene inhibition strategy for cancer gene therapy

NF-κB is a promising target for cancer treatment because of its overactivation in almost all cancers but countless NF-κB inhibitors rarely became clinical drugs due to side effects. In contrast to traditional cancer treatments aimed at inhibiting NF-κB activity, this study develop a novel approach termed HOPE, which focuses on activating the exogenous effector gene CRISPR-Cas13a within cancer cells, achieved by utilizing the NF-κB-specific promoter DMP previously constructed, then targets and suppresses the expression of oncogenes TERT, PLK1, KRAS and MYC at mRNA level. We evaluated the antitumour effects of HOPE in various cultured cells and confirmed it could induce obvious the death of cancer cells without affecting normal cells. By packaging HOPE into adeno-associated virus (AAV) and intravenously injected it to treat mice that were subcutaneously transplanted with colorectal cancer. This validated that rAAV-HOPE could significantly inhibit tumour growth without side effects. Based on the scRNA-seq data, we observed that HOPE could activate the immune system and decrease the proportion of cancer cells, particularly reducing the stemness of cancer cells. This study elucidates an important role of HOPE in inhibiting cancer cell growth both in vitro and in vivo, additionally provides a novel therapeutic technology for cancer gene therapy.

Modern approaches in suicidal gene therapy of malignant neoplasms

One of the promising directions in antitumor therapy is suicidal gene therapy based on the introduction of cytotoxic genes into tumor cells. Most often, these genes encode for enzymes of bacterial or viral origin, capable of direct or indirect killing of tumor cells. This review provides information about modern strategies for suicidal cancer gene therapy, discusses their advantages and disadvantages, and analyzes the properties of a potential candidate for creating a new highly effective suicidal system, combining the advantages of existing approaches.

Nanoliposomes as nonviral vectors in cancer gene therapy.

Nonviral vectors, such as liposomes, offer potential for targeted gene delivery in cancer therapy. Liposomes, composed of phospholipid vesicles, have demonstrated efficacy as nanocarriers for genetic tools, addressing the limitations of off-targeting and degradation commonly associated with traditional gene therapy approaches. Due to their biocompatibility, stability, and tunable physicochemical properties, they offer potential in overcoming the challenges associated with gene therapy, such as low transfection efficiency and poor stability in biological fluids. Despite these advancements, there remains a gap in understanding the optimal utilization of nanoliposomes for enhanced gene delivery in cancer treatment. This review delves into the present state of nanoliposomes as carriers for genetic tools in cancer therapy, sheds light on their potential to safeguard genetic payloads and facilitate cell internalization alongside the evolution of smart nanocarriers for targeted delivery. The challenges linked to their biocompatibility and the factors that restrict their effectiveness in gene delivery are also discussed along with exploring the potential of nanoliposomes in cancer gene therapy strategies by analyzing recent advancements and offering future directions.

Advances and challenges in gene therapy strategies for pediatric cancer: a comprehensive update.

Pediatric cancers represent a tragic but also promising area for gene therapy. Although conventional treatments have improved survival rates, there is still a need for targeted and less toxic interventions. This article critically analyzes recent advances in gene therapy for pediatric malignancies and discusses the challenges that remain. We explore the innovative vectors and delivery systems that have emerged, such as adeno-associated viruses and non-viral platforms, which show promise in addressing the unique pathophysiology of pediatric tumors. Specifically, we examine the field of chimeric antigen receptor (CAR) T-cell therapies and their adaptation for solid tumors, which historically have been more challenging to treat than hematologic malignancies. We also discuss the genetic and epigenetic complexities inherent to pediatric cancers, such as tumor heterogeneity and the dynamic tumor microenvironment, which pose significant hurdles for gene therapy. Ethical considerations specific to pediatric populations, including consent and long-term follow-up, are also analyzed. Additionally, we scrutinize the translation of research from preclinical models that often fail to mimic pediatric cancer biology to the regulatory landscapes that can either support or hinder innovation. In summary, this article provides an up-to-date overview of gene therapy in pediatric oncology, highlighting both the rapid scientific progress and the substantial obstacles that need to be addressed. Through this lens, we propose a roadmap for future research that prioritizes the safety, efficacy, and complex ethical considerations involved in treating pediatric patients. Our ultimate goal is to move from incremental advancements to transformative therapies.

Oncolytic therapy with vaccinia virus carrying IL-24 for hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a highly refractory cancer associated with increasing mortality, which currently lacks effective treatment options. Interleukin-24 (IL-24) is a novel tumor suppressor cytokine that can selectively induce cancer cell apoptosis, and it has been utilized as a cancer gene therapy strategy. The vaccinia virus is a promising strategy for cancer therapy, owing to its direct viral lytic effects, as well as a vehicle to overexpress therapeutic transgenes.MethodsWe constructed a recombinant oncolytic vaccinia viruse (VG9-IL-24) based on vaccinia virus Guang9 (VG9) harboring the IL-24 gene. In vitro, we assessed the replication of VG9-IL-24 in HCC cell lines and normal liver cells and evaluated the cytotoxicity in different cell lines; then, we determined the expression of IL-24 by RT-PCR and ELISA. We examined apoptosis and cell cycle progression in SMMC-7721 cells treated with VG9-IL-24 by flow cytometry. In vivo, we established the SMMC-7721 xenograft mouse model to evaluate the antitumor effects of VG9-IL-24.ResultsIn vitro, VG9-IL-24 efficiently infected HCC cell lines, but not normal liver cells, and resulted in a high level of IL-24 expression and significant cytotoxicity. Moreover, VG9-IL-24 induced an increase in the proportion of apoptotic cells and blocked the SMMC-7721 cell cycle in the G2/M phase. In vivo, tumor growth was significantly suppressed and the survival was prolonged in VG9-IL-24-treated mice.ConclusionsVaccinia virus VG9-mediated gene therapy might be an innovative treatment for cancer with tumor-specific lysis and apoptosis-inducing effects. VG9-IL-24 exhibited enhanced antitumor effects and is a promising candidate for HCC therapy.

Stimuli-Responsive and Highly Penetrable Nanoparticles as a Multifunctional Nanoplatform for Boosting Nonsmall Cell Lung Cancer siRNA Therapy.

In cancer therapy, it is acknowledged that large-size nanoparticles stay in the circulation system for a long time, but their permeability to tumor tissues is poor. To address the conflicting need for prolonging circulation time and favorable tumor tissue penetration ability, a charge conversional multifunctional nanoplatform was strategically designed to improve the efficacy of small interfering RNA (siRNA) therapy against nonsmall cell lung cancer (NSCLC). The development of nanodrug delivery systems (NDDSs) was constructed by loading siRNA on polyamidoamine (PAMAM) dendrimers to build small-sized PAM/siRNA via electrostatic interaction and then capped with a pH-triggered copolymer poly(ethylene glycol) methyl ether (mPEG)-poly-l-lysine (PLL)-2,3-dimethylmaleic anhydride (DMA) (shorted as PLM) under physiological conditions. While in the tumor microenvironment, the acidic reaction of the PLM copolymer changes from negative charge to positive charge due to the cleavable amide bond between mPEG-PLL and DMA, leading to large-size nanoparticles (NPs) with a negative charge that turns into a positive charge and small NPs with a high tumor-penetrating ability. All of the in vitro and in vivo studies validated that PLM/PAM/siRNA NPs possess desirable features including excellent biocompatibility, a prolonged circulation time, significant pH sensitivity, high tumor tissue penetration ability, and sufficient endo-/lysosomal escape. Taken together, all results suggest tremendous potential of the gene therapy based on the stimuli-sensitive PLM/PAM/siRNA NPs, providing a profound application prospective treatment strategy in cancer gene therapy.

Targeting stanniocalcin-1-expressing tumor cells elicits efficient antitumor effects in a mouse model of human lung cancer.

Lung cancer is the most common cause of cancer‐related death in developed countries; therefore, the generation of effective targeted therapeutic regimens is essential. Recently, gene therapy approaches toward malignant cells have emerged as attractive molecular therapeutics. Previous studies have indicated that stanniocalcin‐1 (STC‐1), a hormone involved in calcium and phosphate homeostasis, positively regulates proliferation, apoptosis resistance, and glucose metabolism in lung cancer cell lines. In this study, we investigated if targeting STC‐1 in tumor cells could be a promising strategy for lung cancer gene therapy. We confirmed that STC‐1 levels in peripheral blood were higher in lung cancer patients than in healthy donors and that STC‐1 expression was observed in five out of eight lung cancer cell lines. A vector expressing a suicide gene, uracil phosphoribosyltransferase (UPRT), under the control of the STC‐1 promoter, was constructed (pP STC‐1‐UPRT) and transfected into three STC‐1‐positive cell lines, PC‐9, A549, and H1299. When stably transfected, we observed significant cell growth inhibition using 5‐fluorouracil (5‐FU) treatment. Furthermore, growth of the STC‐1‐negative lung cancer cell line, LK‐2 was significantly arrested when combined with STC‐1‐positive cells transfected with pP STC‐1‐UPRT.We believe that conferring cytotoxicity in STC‐1‐positive lung cancer cells using a suicide gene may be a useful therapeutic strategy for lung cancer.

Inhibition of breast cancer cells by targeting E2F-1 gene and expressing IL15 oncolytic adenovirus.

The wide application of oncolytic adenovirus presents a novel therapeutic strategy for breast cancer gene therapy. Application of adenovirus alone achieves little curative effects on breast cancer. In addition, it is worth exploring the synergistic anti-tumor effect by inserting immunomodulatory factor in oncolytic adenovirus genome. By taking the advantage of the highly proliferative property of breast cancer, a novel recombinant adenovirus which could selectively kill tumor cells is established under an E2F-1 promoter. Also by carrying human Interleukin-15 (IL-15) gene, the oncolytic adenovirus exhibits an immunomodulatory effect. The present study proved that the novel oncolytic virus (SG400-E2F/IL-15) exhibits an enhanced anti-tumor activity both in vitro and in vivo, representing an experimental basis for breast cancer “virus-gene” therapy.

Multifunctional drug carrier based on PEI derivatives loaded with small interfering RNA for therapy of liver cancer

Gene therapy strategies for liver cancer have broad application prospects but still lack a stable and efficient delivery vehicle. To overcome this obstacle, we designed a multifunctional gene delivery vector, sTPssOLP, which was based on oleylamine (OA)-modified disulfide-containing polyethylenimine (PEI) and incorporated into lipids to prepare a lipid nanoparticle. sTPssOLP consisted of the core of PEI derivative and cationic lipids bound to siRNA. The modified polyethylene glycol (PEG) and transferrin (Tf) were partially embedded in the phospholipid bilayer through the lipid and the other as the outer shell. The aim was to use the redox responsiveness of disulfide to trigger siRNA release in cytoplasm to enhance transfection efficiency. Pegylated lipids and Tf focus on increasing cycle life in the body and increasing accumulation at the tumor site of the carrier. In addition, two vectors were prepared as controls, one based on a PEI derivative containing no disulfide bond (POLP) and the other on the surface of the carrier not linked to Tf (PssOLP). PEI derivatives effectively avoid the toxicity problems caused by the use of PEI alone (25 kDa). Meanwhile, it was confirmed by gel retardation experiments that in the presence of dithiothreitol (DTT), the disulfide bond can indeed be reduced and the siRNA entrapped in the vector can be released. Both HepG2 and SMMC had significant uptake of sTPssOLP. The results of intracellular and lysosomal co-localization indicated that sTPssOLP achieved lysosomal escape. RT-PCR and Western blot results also confirmed that sTPssOLP had the best gene silencing activity. In vivo, the tumor inhibition rate of sTPssOLP in nude mice carrying HepG2 xenografts was 56%, which was significantly greater than that of the saline control group. In vivo imaging results showed that fluorescently labeled siRNA loaded in sTPssOLP was able to deliver more to the tumor site. At the same time, it was observed that sTPssOLP did not show significant damage to normal tissues. Therefore, this multifunctional gene delivery vector warrants further investigation.

Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.

Non-small-cell lung cancer homing peptide-labeled dendrimers selectively transfect lung cancer cells.

Lung cancer gene therapies require reagents to selectively transfect lung tumors after systemic administration. We created a reagent called NSCLC-NP by attaching a peptide with binding affinity for lung cancer to polyamidoamine dendrimers. The positively charged dendrimers electrostatically bind negatively charged nucleic acids, inhibit endogenous nucleases and transfect cells targeted by the attached peptide. In vitro, NSCLC-NP complexed to DNA plasmids bound and transfected three human lung cancer cell lines producing protein expression of the plasmid's gene. In vivo, systemically administered NSCLC-NP selectively transfected lung cancer cells growing in RAG1KO mice. The capability of NSCLC-NP to selectively transfect lung cancer allows its future use as a vehicle to implement human lung cancer gene therapy strategies.

Local and Systemic Delivery of Interleukin-12 Gene by Cationic Micelles for Cancer Immunogene Therapy

Immunogene therapy is an alternative strategy for cancer gene therapy. By stimulating the activities of immune cells in the tumor microenvironment, the genetic materials boost the immune response to attack cancer cells resulting in therapeutic effects. Interleukin-12 is an important regulator with great potential in modulating both innate and adaptive immunities. Here, a cancer immunogene therapy strategy was established and evaluated by delivering the IL-12 gene with a novel non-viral gene vector DMP. The DMP cationic micelles were prepared by modifying monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) with the DOTAP lipid via self-assembly. The anti-cancer efficacy of the DMP/IL-12 complex was studied on multiple murine cancer models via both local and systemic administration. Our results demonstrated that the secretory expressed IL-12 cytokine effectively enhanced lymphocytes activities resulting in strong inhibition of cancer cell growth in vitro. Meanwhile, there were obvious tumor regressions achieved in tumor models of C26 colon carcinoma and LL/2 lung cancer in vivo. Multiple anti-cancer mechanisms including T cell infiltration, TNF-α secretion, apoptosis induction and angiogenesis inhibition are involved; no pathology changes were observed in healthy tissues. These results suggest that the DMP/IL-12 complex is a potential candidate for cancer immunogene therapy.

Nonviral Delivery Systems for Cancer Gene Therapy: Strategies and Challenges.

Gene therapy has been receiving widespread attention due to its unique advantage in regulating the expression of specific target genes. In the field of cancer gene therapy, modulation of gene expression has been shown to decrease oncogenic factors in cancer cells or increase immune responses against cancer. Due to the macromolecular size and highly negative physicochemical features of plasmid DNA, efficient delivery systems are an essential ingredient for successful gene therapy. To date, a variety of nanostructures and materials have been studied as nonviral gene delivery systems. In this review, we will cover nonviral delivery strategies for cancer gene therapy, with a focus on target cancer genes and delivery materials. Moreover, we will address current challenges and perspectives for nonviral delivery-based cancer gene therapeutics.

Delivery of interleukin-22 binding protein (IL-22BP) gene by cationic micelle for colon cancer gene therapy.

Gene therapy has provided an alternative strategy for cancer therapy. As an important cytokine, interleukin-22 (IL-22) is not only critical in reinforcing innate immune defenses and tissue regeneration, but also involved in the initial establishment of tumors. A soluble-secreted receptor of the cytokine IL-22, IL-22 binding protein (IL-22BP), binds IL-22 and prevents its binding to the functional transmembrane receptor IL-22R1 complex, inhibiting IL-22-based intracellular cancer proliferation signal. In this work, a novel IL-22BP-based cancer gene therapy strategy was reported for the first time. It was established by delivering IL-22BP gene with a newly developed non-viral gene vector DMP. The DMP cationic micelles were prepared by modifying monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) with DOTAP lipid through self-assembling. The anti-cancer efficacy of the DMP/IL-22BP complex was studied on a colon cancer model by intraperitoneal administration. Our results demonstrated that the secretory expressed IL-22BP cytokine effectively inhibited cancer growth both in vitro and in vivo. Multiple anti-cancer mechanisms including IL-22 blocking, apoptosis inducing, lymphocyte infiltration and angiogenesis inhibition were indicated to be involved while no pathology changes were observed in healthy tissues. These results suggest the DMP/IL-22BP complex to be a potential candidate for cancer gene therapy.

Improving adenoviral vectors and strategies for prostate cancer gene therapy

Gene therapy has been evaluated for the treatment of prostate cancer and includes the application of adenoviral vectors encoding a suicide gene or oncolytic adenoviruses that may be armed with a functional transgene. In parallel, versions of adenoviral vector expressing the p53 gene (Ad-p53) have been tested as treatments for head and neck squamous cell carcinoma and non-small cell lung cancer. Although Ad-p53 gene therapy has yielded some interesting results when applied to prostate cancer, it has not been widely explored, perhaps due to current limitations of the approach. To achieve better functionality, improvements in the gene transfer system and the therapeutic regimen may be required. We have developed adenoviral vectors whose transgene expression is controlled by a p53-responsive promoter, which creates a positive feedback mechanism when used to drive the expression of p53. Together with improvements that permit efficient transduction, this new approach was more effective than the use of traditional versions of Ad-p53 in killing prostate cancer cell lines and inhibiting tumor progression. Even so, gene therapy is not expected to replace traditional chemotherapy but should complement the standard of care. In fact, chemotherapy has been shown to assist in viral transduction and transgene expression. The cooperation between gene therapy and chemotherapy is expected to effectively kill tumor cells while permitting the use of reduced chemotherapy drug concentrations and, thus, lowering side effects. Therefore, the combination of gene therapy and chemotherapy may prove essential for the success of both approaches.

Strategies for Improving siRNA-Induced Gene Silencing Efficiency.

Purpose: Human telomerase reverse transcriptase (hTERT) plays a crucial role in tumorigenesis and progression of cancers. Gene silencing of hTERT by short interfering RNA (siRNA) is considered as a promising strategy for cancer gene therapy. Various algorithms have been devised for designing a high efficient siRNA which is a significant issue in the clinical usage. Thereby, in the present study, the relation of siRNA designing criteria and the gene silencing efficiency was evaluated.Methods: The siRNA sequences were designed and characterized by using on line soft wares. Cationic co-polymer (polyethylene glycol-g-polyethylene imine (PEG-g-PEI)) was used for the construction of polyelectrolyte complexes (PECs) containing siRNAs. The cellular uptake of the PECs was evaluated. The gene silencing efficiency of different siRNA sequences was investigated and the effect of observing the rational designing on the functionality of siRNAs was assessed.Results: The size of PEG-g-PEI siRNA with N/P (Nitrogen/Phosphate) ratio of 2.5 was 114 ± 0.645 nm. The transfection efficiency of PECs was desirable (95.5% ± 2.4%.). The results of Real-Time PCR showed that main sequence (MS) reduced the hTERT expression up to 90% and control positive sequence (CPS) up to 63%. These findings demonstrated that the accessibility to the target site has priority than the other criteria such as sequence preferences and thermodynamic features.Conclusion: siRNA opens a hopeful window in cancer therapy which provides a convenient and tolerable therapeutic approach. Thereby, using the set of criteria and rational algorithms in the designing of siRNA remarkably affect the gene silencing efficiency.

Combined Interleukin 12 and Granulocyte-macrophage Colony-stimulating Factor Gene Therapy Synergistically Suppresses Tumor Growth in the Murine Fibrosarcoma

Background: Interleukin (IL-12) and the granulocyte-monocyte colony-stimulating factor gene (GM-CSF) have been used as immunotherapeutic agents in cancer gene therapy as they activate dendritic cells and boost anti-tumor immune responses. IL-12 and GM-CSF have different roles in anti-tumor immune response. Objectives: The aim of present study was to investigate the anti-tumor effects of combined gene therapy with GM-CSF and IL-12 in a fibrosarcoma mouse model. Methods: To investigate the combined therapeutic effect of GM-CSF and IL-12, WEHI 164 tumor cells were transfected with Murine GM-CSF (m-GM-CSF) and Murine IL-12 (m-IL-12) genes, using Lipofectamine. The fibrosarcoma mouse model was established by subcutaneous injection of transfected cells to Balb/c mice. Mice were sacrificed and the tumors were extracted. Tumor sizes were measured by caliper. The expression of GM-CSF, IL-12 and IFN-γ was studied by real-time PCR and immunoblotting. The expression of Ki-67 (a proliferation marker) in tumor masses was studied by immunohistochemistry staining. Results: The tumor size was reduced in IL-12 + GM-CSF group (P < 0.0001); the results of western blotting and real-time PCR demonstrated that IL-12, GM-CSF and IFN-γ expression increased in IL-12+GM-CSF group (with a relative expression of: 2.86, 1.98, and 2.560). Immunohistochemistry staining indicated that Ki-67 expression was reduced in IL-12 + GM-CSF group. Conclusions: Combination therapy with GM-CSF and IL-12 displayed significant therapeutic effects and represented a promising gene therapy strategy for cancer.

Structural effects and competition mechanisms targeting the interactions between p53 and MDM2 for cancer therapy

Approximately half of all human cancers show normal TP53 gene expression but aberrant overexpression of MDM2 and/or MDMX. This fact suggests a promising cancer therapeutic strategy in targeting the interactions between p53 and MDM2/MDMX. To help realize the goal of developing effective inhibitors to disrupt the p53–MDM2/MDMX interaction, we systematically investigated the structural and interaction characteristics of p53 with inhibitors of its interactions with MDM2 and MDMX from an atomistic perspective using stochastic molecular dynamics simulations. We found that some specific α helices in the structures of MDM2 and MDMX play key roles in their binding to inhibitors, and that the hydrogen bond formed by the Trp23 residue of p53 with its counterpart in MDM2 or MDMX determines the dynamic competition processes of the disruption of the MDM2–p53 interaction and replacement of p53 from the MDM2–p53 complex in vivo. The results reported in this paper are expected to provide basic information for designing functional inhibitors and realizing new strategies of cancer gene therapy.