Synergistic Effects of Temozolomide and Doxorubicin in the Treatment of Glioblastoma Multiforme: Enhancing Efficacy through Combination Therapy.

Glioblastoma multiforme (GBM), a grade IV (WHO classification) malignant brain tumor, poses significant challenges in treatment. The current standard treatment involves surgical tumor removal followed by radiation and chemotherapeutic interventions. However, despite these efforts, the median survival for GBM patients remains low. Temozolomide, an alkylating agent capable of crossing the blood-brain barrier, is currently the primary drug for GBM treatment. Its efficacy, however, is limited, leading to the exploration of combination treatments. In this study, we have investigated the synergistic effects of combining temozolomide with doxorubicin, a chemotherapeutic agent widely used against various cancers. Our experiments, conducted on both temozolomide-sensitive (U87) and -resistant cells (GBM43 and GBM6), have demonstrated a synergistic inhibition of brain cancer cells with this combination treatment. Notably, the combination enhanced doxorubicin uptake and induced higher apoptosis in temozolomide-resistant GBM43 cells. The significance of our findings lies in the potential application of this combination treatment, even in cases of temozolomide resistance. Despite doxorubicin's inability to cross the blood-brain barrier, our results open avenues for alternative delivery methods, such as conjugation with carriers like albumin or local administration at the surgical site through a hydrogel application system. Our study suggests that the synergistic interaction between temozolomide and doxorubicin holds promise for enhancing the efficacy of glioblastoma treatment. The positive outcomes observed in our experiments provide confidence in considering this strategy for the benefit of patients with glioblastoma.

Circumventing Doxorubicin Resistance Using Elastin-like Polypeptide Biopolymer-Mediated Drug Delivery.

Although doxorubicin (dox), an anthracycline antibiotic, is widely used and effective in treating cancer, its treatment efficiency is limited by low blood plasma solubility, poor pharmacokinetics, and adverse side effects, including irreversible cardiotoxicity. Moreover, cancer cells often develop drug resistance over time, which decreases the efficacy of anti-cancer drugs, including dox. In this study, we examine a macromolecular drug delivery system for its ability to specifically deliver doxorubicin to cancer cells with and without drug resistance. This drug delivery system consists of a multi-part macromolecule, which includes the following: elastin-like polypeptide (ELP), cell penetrating peptide (CPP), a cleavable linker (releasing at low pH), and a derivative of doxorubicin. ELP is thermally responsive and improves drug solubility, while the CPP mediates cellular uptake of macromolecules. We compared cytotoxicity of two doxorubicin derivatives, where one is cleavable (DOXO) and contains a pH-sensitive linker and releases dox in an acidic environment, and the other is non-cleavable (ncDox) doxorubicin. Cytotoxicity, apoptosis, cell cycle distribution and mechanism of action of these constructs were tested and compared between dox-responsive MCF-7 and dox-resistant NCI/ADR cell lines. Dox delivered by the ELP construct is comparably toxic to both sensitive and drug resistant cell lines, compared to unconjugated doxorubicin, and given the pharmacokinetic and targeting benefits conveyed by conjugation to ELP, these biopolymers have potential to overcome dox resistance in vivo.

Targeted Drug Delivery Biopolymers Effectively Inhibit Breast Tumor Growth and Prevent Doxorubicin-Induced Cardiotoxicity.

The anticancer agent doxorubicin(dox) has been widely used in the treatment of a variety of hematological malignancies and solid tumors. Despite doxorubicin's efficiency in killing tumor cells, severe damage to healthy tissues, along with cardiotoxicity, limits its clinical use. To overcome these adverse side effects, improve patient safety, and enhance therapeutic efficacy, we have designed a thermally responsive biopolymer doxorubicin carrier that can be specifically targeted to tumor tissue by locally applying mild hyperthermia (41 °C). The developed drug vehicle is composed of the following: a cell penetrating peptide (SynB1) to promote tumor and cellular uptake; thermally responsive Elastin-like polypeptide (ELP); and the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) containing a pH-sensitive hydrazone linker that releases doxorubicin in the acidic tumor environment. We used the in vivo imaging system, IVIS, to determine biodistribution of doxorubicin-delivered ELP in MDA-MB-231 xenografts in nude mice. Tumor bearing mice were treated with a single IV injection of 10 mg/kg doxorubicin equivalent dose with free doxorubicin, thermally responsive SynB1 ELP 1-DOXO, and a thermally nonresponsive control biopolymer, SynB1 ELP 2-DOXO. Following a 2 h treatment with hyperthermia, tumors showed a 2-fold higher uptake when treated with SynB1 ELP 1-DOXO compared to free doxorubicin. Accumulation of the thermally non-responsive control SynB1 ELP2 -DOXO was comparable to free doxorubicin, indicating that an increase in dox accumulation with ELP is due to aggregation in response to thermal targeting. Higher levels of SynB1 ELP1-DOXO and SynB1 ELP2 -DOXO with respect to free doxorubicin were observed in kidneys. Fluorescence intensity from hearts of animals treated with SynB1 ELP1-DOXO show a 5-fold decrease in accumulation of doxorubicin than the same dose of free doxorubicin. SynB1-ELP1-DOXO biopolymers demonstrated a 6-fold increase in tumor/heart ratio in comparison to free doxorubicin, indicating preferential accumulation of the drug in tumors. These results demonstrate that thermally targeted polymers are a promising therapy to enhance tumor targeting and uptake of anticancer drugs and to minimize free drug toxicity in healthy tissues, representing a great potential for clinical application.

Cell-Penetrating Doxorubicin Released from Elastin-Like Polypeptide Kills Doxorubicin-Resistant Cancer Cells in In Vitro Study.

Elastin-like polypeptides (ELPs) undergo a characteristic phase transition in response to ambient temperature. Therefore, it has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be used to target hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of an ELP, a matrix metalloproteinase (MMP) substrate, a cell-penetrating peptide (CPP), and a 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells compared to ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES-SA/Dx5), ELP-released cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells compared to ELP-Dox and free Dox. MMP-digested CPP-Dox showed better membrane penetration and induced more cancer cell death in vitro. This CPP-complexed Dox released from the ELP killed even Dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

Design, Synthesis, and Preliminary Studies of Spiro-isoxazoline-peroxides against Human Cytomegalovirus and Glioblastoma ∥.

The association between glioblastoma (GBM) and human cytomegalovirus (HCMV) infection has been the intensely debated topic over the decades for developing new therapeutic options. In this regard, the peroxides from natural and synthetic sources served as potential antiviral and anticancer agents in the past. Herein, a concise and efficient strategy has been demonstrated to access a novel class of peroxides containing a spiro-isoxazoline to primarily investigate the biological activities. The synthetic compounds were evaluated for in vitro antiviral and antiproliferative activity against HCMV and glioblastoma cell line (GBM6), respectively. While compound 13m showed moderate anti-CMV activity (IC50 = 19 µM), surprisingly, an independent biological assay for compound 13m revealed its antiproliferative activity against the human glioblastoma cell line (GBM6) with an IC50 of 10 µM. Hence, the unification of an isoxazoline and peroxide heterocycles could be a potential direction to initiate the HCMV-GBM drug discovery program.

Evaluation of Elastin-Like Polypeptides for Tumor Targeted Delivery of Doxorubicin to Glioblastoma.

To increase treatment efficiency for glioblastoma, we have developed a system to selectively deliver chemotherapeutic doxorubicin (Dox) to Glioblastoma (GBM) tumors. This carrier is based on elastin-like polypeptide (ELP), which is soluble at physiological temperatures but undergoes a phase transition and accumulates at tumor sites with externally applied, mild (40-41 °C) hyperthermia. The CPP-ELP-Dox conjugate consists of a cell penetrating peptide (CPP), which facilitates transcytosis through the blood brain barrier and cell entry, and a 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensures release of Dox in the lysosomes/endosomes after cellular uptake of the drug conjugate. We have shown that CPP-ELP-Dox effectively inhibits cell proliferation in three GBM cell lines. Both the free drug and CPP-ELP-Dox conjugate exhibited similar in vitro cytotoxicity, although their subcellular localization was considerably different. The Dox conjugate was mainly dispersed in the cytoplasm, while free drug had partial nuclear accumulation in addition to cytoplasmic distribution. The intracellular Dox concentration was increased in the CPP-ELP-Dox cells compared to that in the cells treated with free Dox, which positively correlates with cytotoxic activity. In summary, our findings demonstrate that CPP-ELP-Dox effectively kills GBM cells. Development of such a drug carrier has the potential to greatly improve current therapeutic approaches for GBM by increasing the specificity and efficacy of treatment and reducing cytotoxicity in normal tissues.

Synthesis and Investigation of Novel Spiro-isoxazolines as Anti-Cancer Agents.

A series of structurally diverse 4-bromo spiro-isoxazolines possessing a variety of aromatic and aliphatic substituents at the 3 position, were synthesized through a 1,3-dipolar cycloaddition followed by intramolecular cyclization of a pendant hydroxyl or carboxylic acid group. The biochemical antiproliferative activity was evaluated in vitro by using two breast cancer cell lines (MCF-7 and MDA-MB-231) and two prostate cancer cell lines (PC-3 and DU-145) using the MTT viability assay, and the IC50 values were obtained. Spiro-isoxazoline derivatives bearing a p-chloro or an o-dichloro aromatic substituent at the 3-position of the isoxazoline showed considerable antitumor activities in all four cell lines with IC50 value ranging from 43µM to 56µM.

Polymer-Based Prodrugs: Improving Tumor Targeting and the Solubility of Small Molecule Drugs in Cancer Therapy.

The majority of anticancer drugs have poor aqueous solubility, produce adverse effects in healthy tissue, and thus impose major limitations on both clinical efficacy and therapeutic safety of cancer chemotherapy. To help circumvent problems associated with solubility, most cancer drugs are now formulated with co-solubilizers. However, these agents often also introduce severe side effects, thereby restricting effective treatment and patient quality of life. A promising approach to addressing problems in anticancer drug solubility and selectivity is their conjugation with polymeric carriers to form polymer-based prodrugs. These polymer-based prodrugs are macromolecular carriers, designed to increase the aqueous solubility of antitumor drugs, can enhance bioavailability. Additionally, polymer-based prodrugs approach exploits unique features of tumor physiology to passively facilitate intratumoral accumulation, and so improve chemodrug pharmacokinetics and pharmacological properties. This review introduces basic concepts of polymer-based prodrugs, provides an overview of currently emerging synthetic, natural, and genetically engineered polymers that now deliver anticancer drugs in preclinical or clinical trials, and highlights their major anticipated applications in anticancer therapies.

Effect of basic cell-penetrating peptides on the structural, thermodynamic, and hydrodynamic properties of a novel drug delivery vector, ELP[V5G3A2-150].

Elastin-like polypeptides (ELPs) are large, nonpolar polypeptides under investigation as components of a novel drug delivery system. ELPs are soluble at low temperatures, but they desolvate and aggregate above a transition temperature (TT). This aggregation is being utilized for targeting systemically delivered ELP-drug conjugates to heated tumors. We previously examined the structural, thermodynamic, and hydrodynamic properties of ELP[V5G3A2-150] to understand its behavior as a therapeutic agent. In this study, we investigate the effect that adding basic cell-penetrating peptides (CPPs) to ELP[V5G3A2-150] has on the polypeptide's solubility, structure, and aggregation properties. CPPs are known to enhance the uptake of ELP into cultured cells in vitro and into tumor tissue in vivo. Interestingly, the asymmetric addition of basic residues decreased the solubility of ELP[V5G3A2-150], although below the TT we still observed a low level of self-association that increased with temperature. The ΔH of the aggregation process correlates with solubility, suggesting that the basic CPPs stabilize the aggregated state. This is potentially beneficial as the decreased solubility will increase the fraction aggregated and enhance drug delivery efficacy at a heated tumor. Otherwise, the basic CPPs did not significantly alter the biophysical properties of ELP. All constructs were monomeric at low temperatures but self-associate with increasing temperature through an indefinite isodesmic association. This self-association was coupled to a structural transition to type II ß-turns. All constructs reversibly aggregated in an endothermic reaction, consistent with a reaction driven by the release of water.

The design and delivery of a PKA inhibitory polypeptide to treat SCA1.

Spinocerebellar ataxia-1 (SCA1) is a neurodegenerative disease that primarily targets Purkinje cells (PCs) of the cerebellum. The exact mechanism of PC degeneration is unknown, however, it is widely believed that mutant ataxin-1 becomes toxic because of the phosphorylation of its serine 776 (S776) residue by cAMP-dependent protein kinase A (PKA). Therefore, to directly modulate mutant ATXN1 S776 phosphorylation and aggregation, we designed a therapeutic polypeptide to inhibit PKA. This polypeptide comprised of a thermally responsive elastin-like peptide (ELP) carrier, which increases peptide half-life, a PKA inhibitory peptide (PKI), and a cell-penetrating peptide (Synb1). We observed that our therapeutic polypeptide, Synb1-ELP-PKI, inhibited PKA activity at concentrations similar to the PKI peptide. Additionally, Synb1-ELP-PKI significantly suppressed mutant ATXN1 S776 phosphorylation and intranuclear inclusion formation in cell culture. Further, Synb1-ELP-PKI treatment improved SCA1 PC morphology in cerebellar slice cultures. Furthermore, the Synb1-ELP peptide carrier crossed the blood-brain barrier and localized to the cerebellum via the i.p. or intranasal route. Here, we show the intranasal delivery of ELP-based peptides to the brain as a novel delivery strategy. We also demonstrate that our therapeutic polypeptide has a great potential to target the neurotoxic S776 phosphorylation pathway in the SCA1 disease.

Focused cerebellar laser light induced hyperthermia improves symptoms and pathology of polyglutamine disease SCA1 in a mouse model.

Spinocerebellar ataxia 1 (SCA1) results from pathologic glutamine expansion in the ataxin-1 protein (ATXN1). This misfolded ATXN1 causes severe Purkinje cell (PC) loss and cerebellar ataxia in both humans and mice with the SCA1 disease. The molecular chaperone heat-shock proteins (HSPs) are known to modulate polyglutamine protein aggregation and are neuroprotective. Since HSPs are induced under stress, we explored the effects of focused laser light induced hyperthermia (HT) on HSP-mediated protection against ATXN1 toxicity. We first tested the effects of HT in a cell culture model and found that HT induced Hsp70 and increased its localization to nuclear inclusions in HeLa cells expressing GFP-ATXN1[82Q]. HT treatment decreased ATXN1 aggregation by making GFP-ATXN1[82Q] inclusions smaller and more numerous compared to non-treated cells. Further, we tested our HT approach in vivo using a transgenic (Tg) mouse model of SCA1. We found that our laser method increased cerebellar temperature from 38 to 40 °C without causing any neuronal damage or inflammatory response. Interestingly, mild cerebellar HT stimulated the production of Hsp70 to a significant level. Furthermore, multiple exposure of focused cerebellar laser light induced HT to heterozygous SCA1 transgenic (Tg) mice significantly suppressed the SCA1 phenotype as compared to sham-treated control animals. Moreover, in treated SCA1 Tg mice, the levels of PC calcium signaling/buffering protein calbindin-D28k markedly increased followed by a reduction in PC neurodegenerative morphology. Taken together, our data suggest that laser light induced HT is a novel non-invasive approach to treat SCA1 and maybe other polyglutamine disorders.

Thermally targeted p21 peptide enhances bortezomib cytotoxicity in androgen-independent prostate cancer cell lines.

Prostate cancer remains one of the most common malignancies in men. Besides surgical resection, treatments for prostate cancer include hormone therapy, chemotherapy, and radiation therapy. Advancement of prostate cancer to an androgen-independent state limits the potential of conventional therapeutic approaches. Bortezomib, an FDA-approved proteosomal inhibitor for the treatment of myeloid leukemia, has been shown to have a positive effect on the inhibition of prostate cancer growth. Unfortunately, bortezomib has a very narrow therapeutic window, which can lead to severe side effects. Elastin-like polypeptide (ELP) is a genetically engineered, thermally responsive macromolecular carrier that enables a targeted delivery of the bound molecule because of its soluble property under normal physiologic conditions. In addition, ELP aggregates in response to mild hyperthermia. Using ELP as a carrier, it is possible to improve the pharmacological properties of the therapeutic drug as well as reduce toxicity in normal tissues. In this work, we have investigated the combination treatment of androgen-independent prostate cancer cells with bortezomib and the C-terminal part of the p21(WAF1/CIP1) protein bound to the ELP carrier. We have found that combination treatment with bortezomib and ELP-bound p21(WAF1/CIP1) protein leads to increased cell cycle arrest as well as apoptosis with respect to single treatments. We believe that this approach represents a promising direction for the treatment of androgen-independent prostate cancer.

Structural and hydrodynamic analysis of a novel drug delivery vector: ELP[V5G3A2-150].

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II ß-turn structure, forming a ß-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous ß structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 µM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C.

Targeted c-Myc Inhibition and Systemic Temozolomide Therapy Extend Survival in Glioblastoma Xenografts.

Glioblastoma is a highly aggressive disease with poor patient outcomes despite current treatment options, which consist of surgery, radiation, and chemotherapy. However, these strategies present challenges such as resistance development, damage to healthy tissue, and complications due to the blood-brain barrier. There is therefore a critical need for new treatment modalities that can selectively target tumor cells, minimize resistance development, and improve patient survival. Temozolomide is the current standard chemotherapeutic agent for glioblastoma, yet its use is hindered by drug resistance and severe side effects. Combination therapy using multiple drugs acting synergistically to kill cancer cells and with multiple targets can provide increased efficacy at lower drug concentrations and reduce side effects. In our previous work, we designed a therapeutic peptide (Bac-ELP1-H1) targeting the c-myc oncogene and demonstrated its ability to reduce tumor size, delay neurological deficits, and improve survival in a rat glioblastoma model. In this study, we expanded our research to the U87 glioblastoma cell line and investigated the efficacy of Bac-ELP1-H1/hyperthermia treatment, as well as the combination treatment of temozolomide and Bac-ELP1-H1, in suppressing tumor growth and extending survival in athymic mice. Our experiments revealed that the combination treatment of Bac-ELP1-H1 and temozolomide acted synergistically to enhance survival in mice and was more effective in reducing tumor progression than the single components. Additionally, our study demonstrated the effectiveness of hyperthermia in facilitating the accumulation of the Bac-ELP1-H1 protein at the tumor site. Our findings suggest that the combination of targeted c-myc inhibitory biopolymer with systemic temozolomide therapy may represent a promising alternative treatment option for glioblastoma patients.

In Vitro Comparison Between Ovine and Human Recombinant Hyaluronidase on Hyaluronic Acid Fillers.

Introduction: Injectable hyaluronic acid (HA) fillers are commonly used to provide tissue augmentation and combat the effects of facial aging. Ovine and human recombinant formulations of the enzyme hyaluronidase (HAse) are used interchangeably; however, it is unknown if there exists a difference in their ability to degrade HA. Objective: To compare rates at which ovine and human recombinant forms of HAse degrade various HA fillers in vitro. Methods: Increasing amounts of either ovine or human recombinant HAse were added to fixed amounts of nine unique HA filler products. Degradation rates were then analyzed using a colorimetric method by measuring absorbance levels of degraded product. Results: Human recombinant HAse degraded more HA when compared with ovine HAse overall (p = 0.014, confidence interval [-0.015 to -0.0018]). Conclusions: Human recombinant HAse was found to be more effective on average in degrading HA fillers when compared with ovine HAse in vitro.

A thermally responsive biopolymer conjugated to an acid-sensitive derivative of paclitaxel stabilizes microtubules, arrests cell cycle, and induces apoptosis.

Poor aqueous solubility limits the therapeutic index of paclitaxel as an anti-cancer drug. Synthesis of soluble prodrugs of paclitaxel, or conjugation of the drug to macromolecular carriers have been reported to increase its water-solubility. Macromolecular drug carriers have an added advantage of targeting the drug to the tumor site due to the abnormal tumor blood and lymphatic vasculature. This study describes a thermally responsive macromolecular carrier, elastin-like polypeptide (ELP) for the delivery of paclitaxel. Paclitaxel was bound to ELP by conjugation with the 6-maleimidocaproyl hydrazone derivative of paclitaxel, an acid-sensitive paclitaxel prodrug, for the potential treatment of breast cancer. Focused hyperthermia above a specific transition temperature at the site of a tumor causes ELP to aggregate and accumulate, thereby increasing the local concentration of the drug cargo. The paclitaxel prodrug described here bears an acid-sensitive linker that is cleavable at the lysosomal/endosomal pH, which allows a controlled intracellular release of the drug. The ELP-delivered paclitaxel in the presence of hyperthermia inhibits MCF-7 cell proliferation by stabilizing the microtubule structures, arresting the cells at the G2/M stage, and inducing apoptosis in a manner similar to conventional paclitaxel. It also inhibits proliferation of a paclitaxel resistant MCF-7 cell line. These data provide an in vitro proof of concept for the use of ELP as a delivery vehicle of paclitaxel.

Elastin-like Polypeptide Hydrogels for Tunable, Sustained Local Chemotherapy in Malignant Glioma.

Glioblastoma (GBM) is a primary brain tumor that carries a dismal prognosis, which is primarily attributed to tumor recurrence after surgery and resistance to chemotherapy. Since the tumor recurrence appears near the site of surgical resection, a concept of immediate and local application of chemotherapeutic after initial tumor removal could lead to improved treatment outcome. With the ultimate goal of developing a locally-applied, injectable drug delivery vehicle for GBM treatment, we created elastin-like polypeptide (ELP) hydrogels. The ELP hydrogels can be engineered to release anti-cancer drugs over an extended period. The purpose of this study was to evaluate the biomechanical properties of ELP hydrogels, to characterize their ability to release doxorubicin over time, and to investigate, in vitro, the anti-proliferative effect of Dox-laden ELP hydrogels on GBM. Here, we present microstructural differences, swelling ratio measurements, drug release characteristics, and in vitro effects of different ELP hydrogel compositions. We found that manipulation of the ELP-collagen ratio allows for tunable drug release, that the released drug is taken up by cells, and that incubation with a small volume of ELP-Dox hydrogel drastically reduced survival and proliferation of GBM cells in vitro. These results underscore the potential of ELP hydrogels as a local delivery strategy to improve prognosis for GBM patients after tumor resection.

Inhibition of ovarian cancer cell proliferation by a cell cycle inhibitory peptide fused to a thermally responsive polypeptide carrier.

Current treatment of solid tumors is limited by normal tissue tolerance, resulting in a narrow therapeutic index. To increase drug specificity and efficacy and to reduce toxicity in normal tissues, we have developed a polypeptide carrier for a cell cycle inhibitory peptide, which has the potential to be thermally targeted to the tumor site. The design of this polypeptide is based on elastin-like polypeptide (ELP). The coding sequence of ELP was modified by the addition of the cell penetrating peptide Bac-7 at the N-terminus and a 23 amino acid peptide derived from p21 at the C-terminus (Bac-ELP1-p21). Bac-ELP1-p21 is soluble in aqueous solutions below physiological temperature (37 degrees C) but aggregates when the temperature is raised above 39 degrees C, making it a promising thermally responsive therapeutic carrier that may be actively targeted to solid tumors by application of focused hyperthermia. While Bac-ELP1-p21 at 37 degrees C did not have any effect on SKOV-3 cell proliferation, the use of hyperthermia increased the antiproliferative effect of Bac-ELP1-p21 compared with a thermally unresponsive control polypeptide. Bac-ELP1-p21 displayed both a cytoplasmic and nuclear distribution in the SKOV-3 cells, with nuclear-localized polypeptide enriched in the heated cells, as revealed by confocal microscopy. Using Western blotting, we show that Bac-ELP1-p21 caused a decrease in Rb phosphorylation levels in cells treated at 42 degrees C. The polypeptide also induced caspase activation, PARP cleavage, and cell cycle arrest in S-phase and G2/M-phase. These studies indicate that ELP is a promising macromolecular carrier for the delivery of cell cycle inhibitory peptides to solid tumors.

Thermally Targeted p50 Peptide Inhibits Proliferation and Induces Apoptosis of Breast Cancer Cell Lines.

The application of rationally designed therapeutic peptides (TP) may improve outcomes in cancer treatment. These peptides hold the potential to directly target proliferative pathways and stimulate cell arrest or death pathways. Elastin-like polypeptide (ELP) is an elastin derived biopolymer that undergoes a thermally mediated phase transition. This study employs p50, a nuclear localization sequence derived peptide that inhibits the activation of NFκB and is implicated in cancer cell survival and metastasis. In order to effectively delivery p50, it is conjugated to SynB1-ELP1, a thermally responsive macromolecular carrier. By applying an external heat source, mild hyperthermic conditions (41 °C) induce aggregation and therefore can be used to specifically target ELP to solid tumors in cancer therapy. The addition of a cell penetrating peptide (CPP) to the N-terminus of the macromolecular carrier enhances the cellular uptake and directs the subcellular localization of the bioactive peptide. The novel TP, p50, inhibits proliferation and induces apoptosis of breast cancer cells by blocking the intranuclear import of NFκB. By expanding the repertoire of oncogenic targets, CPPs, and ELP carrier sizes, ELP-based polypeptides may be modulated to optimize the delivery of these novel therapies and allow for the flexibility to create individualized cancer therapies.

Synthesis and biological evaluation of fluoro-substituted spiro-isoxazolines as potential anti-viral and anti-cancer agents.

Electrophilic fluorine-mediated dearomative spirocyclization has been developed to synthesize a range of fluoro-substituted spiro-isoxazoline ethers and lactones. The in vitro biological assays of synthesized compounds were probed for anti-viral activity against human cytomegalovirus (HCMV) and cytotoxicity against glioblastomas (GBM6) and triple negative breast cancer (MDA MB 231). Interestingly, compounds 4d and 4n showed significant activity against HCMV (IC50 ∼ 10 µM), while 4l and 5f revealed the highest cytotoxicity with IC50 = 36 to 80 µM. The synthetic efficacy and biological relevance offer an opportunity to further drug-discovery development of fluoro-spiro-isoxazolines as novel anti-viral and anti-cancer agents.