Gene Therapy Targeting p53 and KRAS for Colorectal Cancer Treatment: A Myth or the Way Forward?

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy worldwide and is responsible as one of the main causes of mortality in both men and women. Despite massive efforts to raise public awareness on early screening and significant advancements in the treatment for CRC, the majority of cases are still being diagnosed at the advanced stage. This contributes to low survivability due to this cancer. CRC patients present various genetic changes and epigenetic modifications. The most common genetic alterations associated with CRC are p53 and KRAS mutations. Gene therapy targeting defect genes such as TP53 (tumor suppressor gene encodes for p53) and KRAS (oncogene) in CRC potentially serves as an alternative treatment avenue for the disease in addition to the standard therapy. For the last decade, significant developments have been seen in gene therapy for translational purposes in treating various cancers. This includes the development of vectors as delivery vehicles. Despite the optimism revolving around targeted gene therapy for cancer treatment, it also has various limitations, such as a lack of availability of related technology, high cost of the involved procedures, and ethical issues. This article will provide a review on the potentials and challenges of gene therapy targeting p53 and KRAS for the treatment of CRC.

Bicistronic transfer of CDKN2A and p53 culminates in collaborative killing of human lung cancer cells in vitro and in vivo.

Cancer therapies that target a single protein or pathway may be limited by their specificity, thus missing key players that control cellular proliferation and contributing to the failure of the treatment. We propose that approaches to cancer therapy that hit multiple targets would limit the chances of escape. To this end, we have developed a bicistronic adenoviral vector encoding both the CDKN2A and p53 tumor suppressor genes. The bicistronic vector, AdCDKN2A-I-p53, supports the translation of both gene products from a single transcript, assuring that all transduced cells will express both proteins. We show that combined, but not single, gene transfer results in markedly reduced proliferation and increased cell death correlated with reduced levels of phosphorylated pRB, induction of CDKN1A and caspase 3 activity, yet avoiding the induction of senescence. Using isogenic cell lines, we show that these effects were not impeded by the presence of mutant p53. In a mouse model of in situ gene therapy, a single intratumoral treatment with the bicistronic vector conferred markedly inhibited tumor progression while the treatment with either CDKN2A or p53 alone only partially controlled tumor growth. Histologic analysis revealed widespread transduction, yet reduced proliferation and increased cell death was associated only with the simultaneous transfer of CDKN2A and p53. We propose that restoration of two of the most frequently altered genes in human cancer, mediated by AdCDKN2A-I-p53, is beneficial since multiple targets are reached, thus increasing the efficacy of the treatment.

A Co-Delivery System of Curcumin and p53 for Enhancing the Sensitivity of Drug-Resistant Ovarian Cancer Cells to Cisplatin.

In order to enhance the sensitivity of drug-resistant ovarian cancer cells to cisplatin (DDP), a co-delivery system was designed for simultaneous delivery of curcumin (CUR) and p53 DNA. Firstly, the bifunctional peptide K14 composed of tumor targeting peptide (tLyP-1) and nuclear localization signal (NLS) was synthesized. A nonviral carrier (PEI-K14) was synthesized by cross-linking low molecular weight polyethyleneimine (PEI) with K14. Then, CUR was coupled to PEI-K14 by matrix metalloproteinase 9 (MMP9)-cleavable peptide to prepare CUR-PEI-K14. A co-delivery system, named CUR-PEI-K14/p53, was obtained by CUR-PEI-K14 and p53 self-assembly. Furthermore, the physicochemical properties and gene transfection efficiency were evaluated. Finally, ovarian cancer cisplatin-resistant (SKOV3-DDP) cells were selected to evaluate the effect of CUR-PEI-K14/p53 on enhancing the sensitivity of drug-resistant cells to DDP. The CUR-PEI-K14/DNA complexes appeared uniformly dispersed and spherical. The particle size was around 20-150 nm and the zeta potential was around 18-37 mV. It had good stability, high transfection efficiency, and low cytotoxicity. CUR-PEI-K14/p53 could significantly increase the sensitivity of SKOV3-DDP cells to DDP, and this effect was better as combined with DDP. The sensitizing effect might be related to the upregulation of p53 messenger RNA (mRNA), the downregulation of P-glycoprotein (P-gp) mRNA, and the upregulation of BCL2-Associated X (bax) mRNA. CUR-PEI-K14/p53 can be used as an effective strategy to enhance the sensitivity of drug-resistant ovarian cancer cells to DDP.

Anti-tumor effect of cisplatin in human oral squamous cell carcinoma was enhanced by andrographolide via upregulation of phospho-p53 in vitro and in vivo.

Oral squamous cell carcinoma is one of the most common neoplasm in the world. Despite the improvements in diagnosis and treatment, the outcome is still poor now. Thus, the development of novel therapeuticapproaches is needed. The aim of this study is to assess the synergistic anti-tumor effect of andrographolide with cisplatin (DDP) in oral squamous cell carcinoma CAL-27 cells in vitro and in vivo. We performed Cell Counting Kit-8 proliferation assay, apoptosis assay, and western blotting on CAL-27 cells treated with andrographolide, DDP or the combination in vitro. In vivo, we also treated CAL-27 xenografts with andrographolide or the combination, and performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and immunohistochemical analysis of Ki-67. The results showed the combination of andrographolide and DDP synergistically inhibited CAL-27 cell proliferation in vitro and caused tumor regression in vivo in the CAL-27 xenografts. In addition, the synergistic anti-tumor effect of andrographolide with synergistic was due to an enhanced apoptosis. Moreover, the combination therapy upregulated the expression level of p-p53 in vitro and decreased Ki-67 expression in vivo. Our data indicate that the combination treatment of andrographolide and DDP results in synergistic anti-tumor growth activity against oral squamous cell carcinoma CAL-27 in vitro and in vivo. These results demonstrated that combination of andrographolide with DDP was likely to represent a potential therapeutic strategy for oral squamous cell carcinoma.

p53 gene therapy-based transarterial chemoembolization for unresectable hepatocellular carcinoma: A prospective cohort study.

BACKGROUND AND AIM: Transarterial chemoembolization (TACE) is used for treating unresectable hepatocellular carcinoma (HCC), but its efficacy still needs to be improved. Recombinant adenovirus p53 (rAd-p53) injection is a gene therapeutic agent that could improve the prognosis of HCC patients. This study aimed to evaluate the efficacy and safety of rAd-p53-based TACE for treating unresectable HCC. METHODS: Prospective analysis of patients who received rAd-p53-based TACE or TACE alone in Chongqing Cancer Institute from January 1, 2011 to December 31, 2012. The primary endpoint is overall survival. The secondary endpoints were progression-free survival, response rate, and safety. RESULTS: One hundred two patients were enrolled in this study. Forty-nine patients received the rAd-p53-based TACE, and 53 patients received TACE alone. The rAd-p53-based TACE treatment strategy improved the overall survival (hazard ratio: 0.58, 95% confidence interval: 0.35-0.96, P = 0.035), progression-free survival (hazard ratio: 0.60, 95% confidence interval: 0.37-0.97, P = 0.037), response rate (P = 0.047) compared with TACE monotherapy. The rAd-p53-based TACE treatment group caused more occurrences of fever than with TACE alone (P = 0.01). However, symptomatic treatment may solve this problem. CONCLUSIONS: rAd-p53-based TACE treatment strategy is effective and safe for treating unresectable HCC. Large-scale randomized clinical trials are needed to verify these results.

Phospho-aspirin (MDC-22) inhibits breast cancer in preclinical animal models: an effect mediated by EGFR inhibition, p53 acetylation and oxidative stress.

BACKGROUND: The anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we synthesized phospho-aspirin (PA-2; MDC-22), a novel derivative of aspirin, and evaluated its chemotherapeutic and chemopreventive efficacy in preclinical models of triple negative breast cancer (TNBC). METHODS: Efficacy of PA-2 was evaluated in human breast cancer cells in vitro, and in orthotopic and subcutaneous TNBC xenografts in nude mice. Mechanistic studies were also carried out to elucidate the mechanism of action of PA-2. RESULTS: PA-2 inhibited the growth of TNBC cells in vitro more potently than aspirin. Treatment of established subcutaneous TNBC xenografts (MDA-MB-231 and BT-20) with PA-2 induced a strong growth inhibitory effect, resulting in tumor stasis (79% and 90% inhibition, respectively). PA-2, but not aspirin, significantly prevented the development of orthotopic MDA-MB-231 xenografts (62% inhibition). Mechanistically, PA-2: 1) inhibited the activation of epidermal growth factor receptor (EGFR) and suppressed its downstream signaling cascades, including PI3K/AKT/mTOR and STAT3; 2) induced acetylation of p53 at multiple lysine residues and enhanced its DNA binding activity, leading to cell cycle arrest; and 3) induced oxidative stress by suppressing the thioredoxin system, consequently inhibiting the activation of the redox sensitive transcription factor NF-κB. These molecular alterations were observed in vitro and in vivo, demonstrating their relevance to the anticancer effect of PA-2. CONCLUSIONS: Our findings demonstrate that PA-2 possesses potent chemotherapeutic efficacy against TNBC, and is also effective in its chemoprevention, warranting further evaluation as an anticancer agent.

Targeted delivery of baicalein-p53 complex to smooth muscle cells reverses pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an uncommon and deadly cardiopulmonary disease. PAH stems essentially from pulmonary artery (PA) remodeling induced predominantly by over-proliferation of PA smooth muscle cells (PASMCs) and inflammation. However, effective treatments are still missing in the clinic because the available drugs consisting of vasodilators are aimed to attenuate PAH symptoms rather than inhibit the remodeling process. Here, we aimed to specifically co-deliver apoptotic executor gene p53 and anti-inflammatory baicalein to PASMCs to alleviate PAH. The targeted co-delivery system was prepared through a carrier-free approach, which was prepared by loading the conjugate, NLS (nuclear localization signal) peptide-p53 gene, onto the baicalein pure crystals, followed by coating with glucuronic acid (GA) for targeting the glucose transport-1 (GLUT-1). The co-delivery system developed has a 200-nm diameter with a rod shape and a drug-loading capacity of 62% (w/w). The prepared system was shown to target PASMCs in vitro and enabled effective gene transfection, efficient apoptosis, and inflammation suppression. In vivo, via targeting the axis lung-PAs-PASMCs, the co-delivery reversed monocrotaline-induced PAH by reducing pulmonary artery pressure, downregulating the proinflammatory cytokine TNF-α, and inhibiting remodeling of both PAs and right ventricular. The potent efficacy may closely correlate with the activation of the signaling axis Bax/Bcl-2/Cas-3. Overall, our results indicate that the co-delivery system holds a significant potential to target the axis of lung-PAs-PASMCs and treat PAH.

Intratumoral delivery of docetaxel enhances antitumor activity of Ad-p53 in murine head and neck cancer xenograft model.

PURPOSE: The aim of this study is to determine the ability of intratumorally delivered docetaxel to enhance the antitumor activity of adenovirus-mediated delivery of p53 (Ad-p53) in murine head and neck cancer xenograft model. MATERIALS AND METHODS: A xenograft head and neck squamous cell carcinoma mouse model was used. Mice were randomized into 4 groups of 6 mice receiving 6 weeks of biweekly intratumoral injection of (a) diluent, (b) Ad-p53 (1 x 10(10) viral particles per injection), (c) docetaxel (1 mg/kg per injection), and (d) combination of Ad-p53 (1 x 10(10) viral particles per injection) and docetaxel (1 mg/kg per injection). Tumor size, weight, toxicity, and overall and disease-free survival rates were determined. RESULTS: Intratumoral treatments with either docetaxel alone or Ad-p53 alone resulted in statistically significant antitumor activity and improved survival compared with control group. Furthermore, combined delivery of Ad-p53 and docetaxel resulted in a statistically significant reduction in tumor weight when compared to treatment with either Ad-p53 or docetaxel alone. CONCLUSION: Intratumoral delivery of docetaxel enhanced the antitumor effect of Ad-p53 in murine head and neck cancer xenograft model. The result of this preclinical in vivo study is promising and supports further clinical testing to evaluate efficacy of combined intratumoral docetaxel and Ad-p53 in treatment of head and neck cancer.

Investigation on the Genomic Characterization of Uterine Sarcoma for rAd-p53 Combined with Chemotherapy Treatment.

The aim is to investigate the genomic characterization of uterine sarcoma for rAd-p53 (Gendicine®) combined with chemotherapy treatment. We recently published an article on 12 cases of uterine sarcomas, which were treated with rAd-p53 combined with chemotherapy. We found that rAd-p53 combined with chemotherapy is effective for various uterine sarcomas. Pretreatment pathological specimens of four uterine sarcoma patients were collected from the above recent clinical research and numbered 1-4A/B. Tumor samples were subjected to targeted sequencing by using a 416 genes panel. We profiled the mutation spectrum and tumor mutation burden in the tumors, identified mutated genes, and explored their gene function. We also verified the p53 protein expression using immunohistochemistry. We identified a total of 30 mutated genes that were found from the next-generation sequencing test results. The average number of mutated genes was up to seven in the five samples. TP53 gene was mutated in two of the four patients, No. 1 and No. 4B. They are c.C833G (p.P278R) missense mutation and a point mutation (C141*) that result in a premature stop codon. We did not find a mutated TP53 gene in the other two cases, but we identified mutated genes, including CREBBP, LYN, CDKN2A, and JAK2, which were located upstream of the TP53 gene; they may have an impact on TP53. We also identified 11 additional genes which are involved in p53-related signaling pathways or have interaction with p53. Compared to solid tumor mutational burden (TMB) distribution, none of their TMB was ranking in the top 25%. Mutant p53 protein expression was positive in two specimens. Our results demonstrated that the TP53 signaling pathway plays an important role in uterine sarcoma tumorigenesis. TP53 and the upstream genes such as CREBBP, LYN, CDKN2A, and JAK2 may be involved in the genomic characterization for rAd-p53 (Gendicine) combined with chemotherapy in uterine sarcoma. Besides, the average amount of mutated genes from every patient is large.

Evaluation of safety and efficacy of p53MVA vaccine combined with pembrolizumab in patients with advanced solid cancers.

BACKGROUND: Vaccination of cancer patients with p53-expressing modified vaccinia Ankara virus (p53MVA) has shown in our previous studies to activate p53-reactive T cells in peripheral blood but without immediate clinical benefit. We hypothesized that the immunological responses to p53MVA vaccine may require additional immune checkpoint blockade to achieve clinically beneficial levels. We therefore conducted a phase I trial evaluating the combination of p53MVA and pembrolizumab (anti-PD-1) in patients with advanced solid tumors. PATIENTS AND METHODS: Eleven patients with advanced breast, pancreatic, hepatocellular, or head and neck cancer received up to 3 triweekly vaccines in combination with pembrolizumab given concurrently and thereafter, alone at 3-week intervals until disease progression. The patients were assessed for toxicity and clinical response. Correlative studies analyzed p53-reactive T cells and profile of immune function gene expression. RESULTS: We observed clinical responses in 3/11 patients who remained with stable disease for 30, 32, and 49 weeks. Two of these patients showed increased frequencies and persistence of p53-reactive CD8+ T cells and elevation of expression of multiple immune response genes. Borderline or undetectable p53-specific T cell responses in 7/11 patients were related to no immediate clinical benefit. The first study patient had a grade 5 fatal myocarditis. After the study was amended for enhanced cardiac monitoring, no additional cardiac toxicities were noted. CONCLUSION: We have shown that the combination of p53MVA vaccine with pembrolizumab is feasible, safe, and may offer clinical benefit in select group of patients that should be identified through further studies.

Delivery of proteins encapsulated in chitosan-tripolyphosphate nanoparticles to human skin melanoma cells.

We have successfully encapsulated two proteins, bovine serum albumin (BSA) and p53, in chitosan-tripolyphosphate (TPP) nanoparticles at various pH values from 5.5 to 6.5 and delivered the particles to human melanoma cells. The particles have diameters ranging from 180 nm to 280 nm and a zeta potential of +15 to + 40 mV. Cellular uptake of the particles by human skin melanoma cells was evaluated by: (i) fluorescence microscopy and (ii) gel electrophoresis showing that FITC-labeled BSA and p53 could be recovered in the soluble cell fraction after lysis of the cells. Our data also show that the highest cellular uptake takes place at the lowest pH as the particles have the highest positive charge under these conditions. The method we describe appears to be a general method for delivery of proteins to cells using chitosan-TPP nanoparticles as a drug delivery system, since structurally unrelated proteins such as BSA and p53 with different isoelectrical points can be encapsulated in the chitosan-TPP nanoparticles and be effectively internalized by the cells.

Redox-responsive polymer inhibits macrophages uptake for effective intracellular gene delivery and enhanced cancer therapy.

The development of advanced gene delivery carriers with stimuli-responsive release manner for tumor therapeutics is desirable, since they can exclusively release the therapeutic gene via their structural changes in response to the specific stimuli of the target site. Moreover, interactions between macrophages and drug delivery systems (DDSs) seriously impair the treatment efficiency of DDSs, thus macrophages uptake inhibition would to some extent improve the intracellular uptake of DDSs in tumor cells. Herein, a PEGylated redox-responsive gene delivery system was developed for effective cancer therapy. PEG modified glycolipid-like polymer (P-CSSO) was electrostatic interacted with p53 to form P-CSSO/p53 complexes, which exhibited an enhanced redox sensitivity in that the disulfide bond was degraded and the rate the plasmid released from P-CSSO was 2.29-fold that of nonresponsive platform (P-CSO-SA) in 10 mM levels of glutathione (GSH). PEGylation could significantly weaken macrophages uptake, while enhance the accumulation of P-CSSO in tumor cells both in vitro and in vivo. Compared with nonresponsive complexes (P-CSO-SA/p53) (59.2%) and Lipofectamine™ 2000/p53 complexes (52.0%), the tumor inhibition rate of P-CSSO/p53 complexes (77.1%) significantly increased, which was higher than CSSO/p53 complexes (69.9%). The present study indicates that tumor microenvironment sensitive and macrophages uptake suppressive P-CSSO/p53 is a powerful in vivo gene delivery system for enhanced anticancer therapy.

Zinc cooperates with p53 to inhibit the activity of mitochondrial aconitase through reactive oxygen species accumulation.

Metabolic reprogramming is a central hallmark of cancer. Therefore, targeting metabolism may provide an effective strategy for identifying promising drug targets for cancer treatment. In prostate cancer, cells undergo metabolic transformation from zinc-accumulating, citrate-producing cells to citrate-oxidizing malignant cells with lower zinc levels and higher mitochondrial aconitase (ACO2) activity. ACO2 is a Krebs cycle enzyme that converts citrate to isocitrate and is sensitive to reactive oxygen species (ROS)-mediated damage. In this study, we found that the expression of ACO2 is positively correlated with the malignancy of prostate cancer. Both zinc and p53 can lead to an increase in ROS. ACO2 can be a target for remodeling metabolism by sensing changes in the ROS levels of prostate cancer. Our results indicate that targeting ACO2 through zinc and p53 can change prostate cancer metabolism, and thus provides a potential new therapeutic strategy for prostate cancer.

Co-delivery of p53 and MDM2 inhibitor RG7388 using a hydroxyl terminal PAMAM dendrimer derivative for synergistic cancer therapy.

P53 inactivation is often achieved through gene mutation and the excessive activity of its major negative regulator, murine double minute 2 protein (MDM2). In the present study we utilized a PAMAM-OH derivative (PAMSPF) to co-deliver p53 plasmid and MDM2 inhibitor (RG7388) to the tumor site and evaluated the synergistic anti-tumor effect of p53 plasmid and RG7388. PAMSPF was able to condense DNA and encapsulate RG7388 to form spherical nanoparticles (PAMSPF/p53/RG) with particle sizes of around 200 nm, and remain stable in the presence of heparin and nuclease. The drug loading capacity and encapsulation efficiency of RG7388 in PAMSPF/p53/RG were 0.5% and 92.5%, respectively. The p53 expressions in MDA-MB-435, p53-wild type MCF-7 cells (MCF-7/WT) and p53-silenced MCF-7 cells (MCF-7/S) treated with PAMSPF/p53/RG were promoted significantly. As a result, PAMSPF/p53/RG was able to inhibit cell proliferation, arrest cell cycle, and induce cell apoptosis of MDA-MB-435, MCF-7/WT and MCF-7/S cells. PAMSPF/p53/RG suppressed human umbilical vascular endothelial cells (HUVECs) migration, invasion and tube formation through decreasing the VEGF expression. And the biological activities described above of PAMSPF/p53/RG were significantly higher than those of PAMSPF/53 and PAMSPF/RG, exhibiting the synergistic actions of p53 plasmid and RG7388. In addition, intravenous administration of PAMPSF/p53/RG inhibited tumor growth of MDA-MB-435 and MCF-7/WT xenograft mice models, and induced no substantial weight loss. PAMSPF/p53/RG also reduced cell proliferation, and induced cell apoptosis in vivo based on the immunohistochemistry results. Collectively, PAMSPF/p53/RG is an excellent system for gene and drug co-delivery, and the combined treatment of p53 plasmid and RG7388 possesses a synergistic antitumor activity both in vitro and in vivo. STATEMENT OF SIGNIFICANCE: In the present study we utilized a PAMAM-OH derivative (PAMSPF) to co-deliver p53 plasmid and RG7388 (MDM2 inhibitor) and evaluated their synergistic anti-tumor effect. PAMSPF could condense p53 plasmid and encapsulate RG7388 to form nanoparticles (PAMSPF/p53/RG). The p53 expressions in MDA-MB-435, p53-wild type MCF-7 cells (MCF-7/WT) and p53-silenced MCF-7 cells (MCF-7/S) treated with PAMSPF/p53/RG were promoted significantly. As a result, PAMSPF/p53/RG could inhibit cell proliferation, arrest cell cycle, and induce cell apoptosis of three kinds of cells. In addition, intravenous administration of PAMPSF/p53/RG inhibited tumor growth of MDA-MB-435 and MCF-7/WT xenograft mice models. Collectively, PAMSPF/p53/RG is an excellent system for gene and drug co-delivery, and the combined treatment of p53 plasmid and RG7388 possesses a synergistic antitumor activity.

ARMMs as a versatile platform for intracellular delivery of macromolecules.

Majority of disease-modifying therapeutic targets are restricted to the intracellular space and are therefore not druggable using existing biologic modalities. The ability to efficiently deliver macromolecules inside target cells or tissues would greatly expand the current landscape of therapeutic targets for future generations of biologic drugs, but remains challenging. Here we report the use of extracellular vesicles, known as arrestin domain containing protein 1 [ARRDC1]-mediated microvesicles (ARMMs), for packaging and intracellular delivery of a myriad of macromolecules, including the tumor suppressor p53 protein, RNAs, and the genome-editing CRISPR-Cas9/guide RNA complex. We demonstrate selective recruitment of these macromolecules into ARMMs. When delivered intracellularly via ARMMs, these macromolecules are biologically active in recipient cells. P53 delivered via ARMMs induces DNA damage-dependent apoptosis in multiple tissues in mice. Together, our results provide proof-of-principle demonstration that ARMMs represent a highly versatile platform for packaging and intracellular delivery of therapeutic macromolecules.

[Treatment of mouse liver metastasis by intraportal injection of Adv-p53].

OBJECTIVE: To investigate the anti-tumor effect of intraportal administration of Adv-p53 in the treatment of the liver metastasis in mice. METHODS: 2 x 10(5) of MCA-205 cells were injected into the mouse portal vein to establish a murine liver metastasis model. The spleen was transpositioned subcutaneously to enable the administration of Adv-p53 continually into the portal system. Different doses of Adv-p53 were injected intraportally, while HBSS and Adv-CMV were injected intraportaly in the control group. Tumors in the liver were examined on day 21 after Adv-p53 administration. RESULTS: The liver weight in the Adv-p53 treated mice on day 0 group (1.20 +/- 0.34 g) was significantly less than that in the Adv-CMV group (2.59 +/- 0.48 g, P < 0.05). The number of metastatic nodules in the Adv-p53 treated mice on day 0 group (9.0 +/- 9.9) was significantly less than that in the Adv-CMV group (57.1 +/- 11.3, P < 0.05), indicating that intraportal administration of Adv-p53 inhibited the formation of liver metastasis. This anti-tumor effect was in a dose-dependent manner. After the liver metastasis was formed, Adv-p53 was administered intraportally. The liver weight in the Adv-p53 treated mice on day 5 group (1.22 +/- 0.09 g) was significantly less than that in the Adv-CMV group (3.98 +/- 1.01 g , P < 0.05). The number of metastatic nodules in the Adv-p53 treaed mice on day 5 group (5.5 +/- 3.5) was significantly less than that in the Adv-CMV group (113.2 +/- 5.8, P < 0.05). Repeatedly intraportal administration of Adv-p53 could enhance this anti-tumor effect. CONCLUSION: Local administration of Adv-p53 into the portal system would be a useful strategy for the liver metastasis treatment.

Synergy between Paclitaxel and Anti-Cancer Peptide PNC-27 in the Treatment of Ovarian Cancer.

OBJECTIVES: Paclitaxel is widely used in the treatment of gynecologic malignancies. It targets tumor cells in the M phase of the cell cycle. Cells in other phases survive the insult and repopulate the tumor. PNC-27 is a peptide synthesized of amino acids of the p53-MDM-2 binding domain. It kills various cancer cell lines in a dose-dependent manner. The goal of this study is to assess ovarian cancer cells' sensitivity to PNC-27 after surviving exposure to paclitaxel and to investigate the potential for synergy between PNC-27 and paclitaxel in the treatment of ovarian cancer. METHODS: The impact of exposure to paclitaxel on the surface expression of MDM-2 was assessed with the use of flow cytometry. For measurement of cytotoxicity in vitro, ID8 cells were exposed to paclitaxel for 12 hours in various concentrations. At 12 hours, the drug containing media was removed and the cells were cultured in media containing various concentrations of PNC-27 for 24 hours. Viability was assessed with the use of an MTT assay. Survival fractions were plotted against drug concentrations and the data were fit to logistic dose-response curves. Isoeffective combinations were used to create isobolograms. The combined treatment with weekly paclitaxel and PNC-27 was tested in an intraperitoneal mouse model of ovarian cancer (ID8). RESULTS: Exposure to paclitaxel rendered incomplete time-dependent killing, while PNC-27 mediated comprehensive, dose-dependent killing of ID8 cells. The cytotoxic effect of PNC-27 was dependent on its binding to MDM-2. Blocking MDM-2 inhibited the killing by PNC-27. ID8 cells surviving paclitaxel demonstrated increased expression of MDM-2 and increased susceptibility to PNC-27. Isobologram for dose combinations that were isoeffective indicates synergistic effect between the 2 agents (Combination index <1). In an in vivo model of ovarian cancer (ID8), the addition of PNC-27 to weekly paclitaxel administration significantly reduces tumor growth. CONCLUSIONS: These data demonstrate synergism between PNC-27 and paclitaxel. PNC-27 could target cells surviving paclitaxel and improve its antitumor effect.

Combination therapy with the farnesyl protein transferase inhibitor SCH66336 and SCH58500 (p53 adenovirus) in preclinical cancer models.

SCH66336 is a p.o.-active, farnesyl protein transferase inhibitor. SCH66336 inhibits farnesylation of RAS and other proteins in tumor cells and suppresses tumor growth in human xenograft and transgenic mouse cancer models in vivo. SCH58500 is a replication-deficient, recombinant adenovirus, which expresses the human p53 tumor suppressor. In preclinical models, SCH58500 has therapeutic efficacy against a wide range of human tumor types containing nonfunctional p53 and enhanced activity in combination with many chemotherapeutic drugs. Here we report that combination therapy with SCH66336 and SCH58500 has synergistic or additive antiproliferative effects on a panel of tumor cells lines in vitro. The efficacy of the three-drug combination of SCH66336, SCH58500, and paclitaxel was also examined in vitro. Each two-drug interaction displayed such marked synergy, the addition of a third drug to the statistical model could only yield additivity. Greater combined efficacy for SCH66336 and SCH58500 was also observed in vivo in the DU-145 human prostate and wap-ras/F transgenic mouse cancer models.

Evaluation of efficacy and safety for recombinant human adenovirus-p53 in the control of the malignant pleural effusions via thoracic perfusion.

A certain number of studies have showed that p53 gene transfer has an anti-tumor activity in vitro and in vivo. This study was to evaluate the efficacy and safety of thoracic perfusion of recombinant human adenovirus p53 (rAd-p53, Gendicine) for controlling malignant pleural effusion (MPE). We searched for the relevant studies from the database of MEDLINE, Web of Science, EMBASE, Cochrance Library and CNKI to collect the trials concerning the efficacy and safety of rAd-p53 to treat MPE. Fourteen randomised controlled trials (RCTs) with 879 patients were involved in this analysis. The rAd-p53 combined with chemotherapeutic agents significantly improved the overall response rate (ORR) (P < 0.001; odds ratio = 3.73) and disease control rate (DCR) (P < 0.001; odds ratio = 2.32) of patients with MPE as well as the quality of life (QOL) of patients (P < 0.001; odds ratio = 4.27), compared with that of chemotherapeutic agents alone. In addition, the participation of rAd-p53 did not have an obvious impact on the most of incidence of adverse reactions (AEs) (P < 0.05) except the fever (P < 0.001). However, the fever was self-limited and could be tolerated well. The application of rAd-p53 through thoracic perfusion for treating MPE had a better efficacy and safety, which could be a potential choice for controlling MPE.

Novel luminescent silica nanoparticles (LSN): p53 gene delivery system in breast cancer in vitro and in vivo.

OBJECTIVES: Mutations in the p53 tumor suppressor gene are one among the most common genetic abnormalities to be described in breast cancer. However, there are a few recant reports on non-viral vector-mediated p53 gene delivery in breast cancer. METHODS: A new formulation of luminescent silica nanoparticles (LSNs) for gene delivery was produced by the two-step method with slight modification. KEY FINDINGS: The pp53 plasmid constructs (p53-EGFP)/LSNs complexes were transfected into human breast cancer cell (MCF-7) and transfection efficiency was determined by FACS analysis. The gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis respectively. Further the growth inhibition through induced apoptosis with pp53-EGFP/LSNs complex were assessed by trypan blue exclusion assay and annexin V staining, respectively. Interestingly the in vivo biodistribution of plasmid DNA study revealed the occurrence was investigated by PCR and RT-PCR. The transfection efficiency of LSNs showed the highest transfection efficiency among the LSN formulation was higher than that of commercially available Lipofectin®. The LSNs-mediated transfection of the p53 gene resulted in efficient high level of wild-type p53 mRNA and protein expression levels in MCF-7 cells. Selected tissues were analyzed for any potential toxicity by histological analysis the efficient reestablishment of wild-type p53 function in breast cancer cells restored the p53 dependent apoptotic pathway. CONCLUSIONS: Taken together, our results reveal that cationic LSN-mediated p53 gene delivery may have potential application as a non-viral vector-mediated breast cancer gene therapy due to its effective induction of apoptosis and tumor growth inhibition.