PEtOx-DOPE nanoliposomes functionalized with peptide 563 in targeted BikDDA delivery to prostate cancer.

Background: Nanocarrier-based systems have cultivated significant improvements in prostate cancer therapy. However, the efforts are still limited in clinical applicability, and more research is required for the development of effective strategies. Here, we describe a novel nanoliposomal system for targeted apoptotic gene delivery to prostate cancer. Methods: Poly (2-ethyl-2-oxazoline) (PEtOx) dioleoyl phosphatidylethanolamine (DOPE) nanoliposomes were conjugated with the prostate-specific membrane antigen (PSMA)-targeting peptide GRFLTGGTGRLLRIS (P563) and loaded with BikDDA, a mutant form of the proapoptotic Bik. We selected 22Rv1 cells with moderate upregulation of PSMA to test the in vitro uptake, cell death, and in vivo anticancer activity of our formulation, P563-PEtOx-DOPE-BikDDA. Results: BikDDA was upregulated in 22Rv1 cells, inducing cell death, and CD-1 nude mice xenografts administered with the formulation showed significant tumor regression. Conclusion: We suggest that P563-PEtOx-DOPE-BikDDA nanoliposomes can serve as prominent gene carriers against prostate cancer.

A Novel PEtOx-Based Nanogel Targeting Prostate Cancer Cells for Drug Delivery.

This study focuses on creating a specialized nanogel for targeted drug delivery in cancer treatment, specifically targeting prostate cancer. This nanogel (referred to as SGK 636/Peptide 563/PEtOx nanogel) is created using hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx) through a combination of living/cationic ring-opening polymerization (CROP) and alkyne-azide cycloaddition (CuAAC) "click" chemical reactions. A fluorescent probe (BODIPY) is also conjugated with the nanogel to monitor drug delivery. The characterizations through 1 H-NMR, and FT-IR, SEM, TEM, and DLS confirm the successful production of uniform, and spherical nanogels with controllable sizes (100 to 296 nm) and stability in physiological conditions. The biocompatibility of nanogels is evaluated using MTT cytotoxicity assays, revealing dose-dependent cytotoxicity. Drug-loaded nanogels exhibited significantly higher cytotoxicity against cancer cells in vitro compared to drug-free nanogels. Targeting efficiency is examined using both peptide-conjugated and peptide-free nanogels, with the intracellular uptake of peptide 563-conjugated nanogels by tumor cells being 60-fold higher than that of nanogels without the peptide. The findings suggest that the prepared nanogel holds great potential for various drug delivery applications due to its ease of synthesis, tunable functionality, non-toxicity, and enhanced intracellular uptake in the tumor region.

Targeting prostate cancer with docetaxel-loaded peptide 563-conjugated PEtOx-co-PEI30%-b-PCL polymeric micelle nanocarriers.

Prostate cancer is a global disease that negatively affects the quality of life. Although various strategies against prostate cancer have been developed, only a few achieved tumor-specific targeting. Therefore, a special emphasis has been placed on the treatment of cancer using nano-carrier-encapsulated chemotherapeutic agents conjugated with tumor-homing peptides. The targeting strategy coupling the drugs with nanotechnology helps to overcome the most common barriers, such as high toxicity and side effects. Prostate-specific membrane antigen has emerged as a promising target molecule for prostate cancer and shown to be targeted with high affinity by GRFLTGGTGRLLRIS peptide known as peptide 563 (P563). Here, we aimed to assess the in vitro and in vivo targeting efficiency, safety, and efficacy of P563-conjugated, docetaxel (DTX)-loaded polymeric micelle nanoparticles (P563-PEtOx-co-PEI30%-b-PCL-DTX) against prostate cancer. To this end, we analyzed the cytotoxic activity of P563-PEtOx-co-PEI30%-b-PCL and P563-PEtOx-co-PEI30%-b-PCL-DTX by a cell proliferation assay using PNT1A and 22Rv1 cells. We have also determined the targeting selectivity of P563-PEtOx-co-PEI30%-b-PCL-FITC by flow cytometry and assessed the induction of cell death by western blot and TUNEL assays for P563-PEtOx-co-PEI30%-b-PCL-DTX in 22Rv1 cells. To investigate the in vivo efficacy, we administered DTX in the free form or in polymeric micelle nanoparticles to athymic CD-1 nu/nu mice 22Rv1 xenograft models and performed histopathological analyses. Our study showed that targeting prostate cancer with P563-conjugated PEtOx-co-PEI30%-b-PCL polymeric micelles could exert a potent anti-cancer activity with low side effects.

Palbociclib negatively regulates fatty acid synthesis due to upregulation of AMPKα and miR-33a levels to increase apoptosis in Panc-1 and MiaPaCa-2 cells.

Fatty acids (FAs) synthesis mechanism has various regulators such as fatty acid synthase (FASN), AMP-regulated protein kinase (AMPK), or mammalian target of rapamycin (mTOR), which are aberrantly dysregulated in various pancreatic cancer cells. In this study, we aim to understand the regulatory role of palbociclib, a CDK4/6 inhibitor, on the cellular energy metabolism through regulation of AMPK/mTOR signaling by modulation of intracellular miR-33a levels in Panc-1 and MiaPaCa-2 cells. Palbociclib downregulated FAs metabolism more effectively in MiaPaCa-2 cells than Panc-1 cells. Moreover, palbociclib treatment increased the levels of miR-33a in each cell line albeit a higher increase was evident in MiaPaCa-2 cells. Stress-mediated activation of mTOR signaling axis was found associated with palbociclib-mediated AMPKα activation and miR33a upregulation. These findings provided that a deeper understanding about possible interactions of cell cycle activity and reduction of FAs synthesis may facilitate the enhancement of cell death mechanisms in pancreatic cancer cells.

Nitrosylation of tissue transglutaminase enhances fibroblast migration and regulates MMP activation.

In wound healing, the TG2 enzyme plays a dual functional role. TG2 has been shown to regulate extracellular matrix (ECM) stabilization by its transamidase activity while increasing cell migration by acting as a cell adhesion molecule. In this process, nitric oxide (NO) plays a particularly important role by nitrosylation of free cysteine ​​residues on TG2, leading to the irreversible inactivation of the catalytic activity. In this study, transfected fibroblasts expressing TG2 under the control of the tetracycline-off promoter were treated with NO donor S-nitroso-N-acetyl penicillamine (SNAP) to analyze the interplay between NO and TG2 in the regulation of cell migration/invasion as well as TGF-ß1-dependent MMP activation. Our results demonstrated that inhibition of TG2 cross-linking activity by SNAP promoted the migration and invasion capacity of fibroblasts by hindering TG2-mediated TGF-ß1 activation. While the inhibition of TG2 activity by NO downregulated the biosynthesis and activity of MMP-2 and MMP-9, that of MMP-1a and MMP-13 was shown to be upregulated in a TGF-ß1-dependent manner under the same conditions. In the presence of SNAP, interaction of TG2 with its cell surface binding partners Integrin-ß1 and Syndecan-4 was reduced, which was paralleled by an increase in TG2 and PDGF association. These findings suggests that migratory phenotype of fibroblasts can be regulated by the interplay between nitric oxide and TG2 activity.

Delivery of doxorubicin loaded P18 conjugated-poly(2-ethyl-oxazoline)-DOPE nanoliposomes for targeted therapy of breast cancer.

Breast cancer, a heterogeneous disease, has the highest incidence rate and is a major cause of death in females worldwide. Drug delivery by using nanotechnology has shown great promise for improving cancer treatment. Nanoliposomes are known to have enhanced accumulation ability in tumors due to prolonged systemic circulation. Peptide 18 (P18), a tumor homing peptide targeting keratin-1 (KRT-1), was previously shown to have high binding affinity towards breast cancer cells. In this study, we investigate the ability of P18 conjugated PEtOx-DOPE nanoliposomes (P18-PEtOx-DOPE) for the targeted delivery of doxorubicin to AU565 breast cancer model. Toxicology studies of PEtOx-DOPE nanoliposomes performed on normal breast epithelial cells (MCF10A), showed minimal toxicity. Doxorubicin delivery by P18-PEtOx-DOPE to AU565 cells induces cytotoxicity in a dose and time dependent manner causing mitotic arrest in G2/M phase at 24 h. Anti-cancer activity of P18-PEtOx-DOPE-DOX nanoliposomes on AU565 cells was detected by Annexin V/PI apoptosis assay. In terms of in vivo antitumor efficacy, P18-PEtOx-DOPE-DOX nanoliposomes administration to AU565 CD-1 nu/nu mice model showed significant decrease in tumor volume suggesting that DOX delivered by these nanoliposomes elicited a strong antitumor response comparable to the free delivery of doxorubicin. Overall, our results offered preclinical proof for the use of P18-PEtOx-DOPE-DOX nanoliposomes in KRT-1+ breast cancer therapy.

Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice.

The SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A and smallpox proved to be reliable approaches for immunization for prolonged periods. In this study, a gamma-irradiated inactivated virus vaccine does not require an extra purification process, unlike the chemically inactivated vaccines. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral RNA copy per dose) of OZG-38.61.3 was initially determined in BALB/c mice. This was followed by testing the immunogenicity and protective efficacy of the vaccine. Human ACE2-encoding transgenic mice were immunized and then infected with the SARS-CoV-2 virus for the challenge test. This study shows that vaccinated mice have lowered SARS-CoV-2 viral RNA copy numbers both in oropharyngeal specimens and in the histological analysis of the lung tissues along with humoral and cellular immune responses, including the neutralizing antibodies similar to those shown in BALB/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.

Garlic (Allium sativum)-derived SEVs inhibit cancer cell proliferation and induce caspase mediated apoptosis.

As a key component of the cell-to-cell communication, small extracellular vesicles (SEVs) released from various sources are known to be affecting the physiological conditions of the target cells. Although it has been suggested that edible plant-derived nanoparticles contributes to the cross kingdom communication with the mammalian cells, the effect of these particles on cancer cell progression still needs a further exploration. Here, we isolated and then characterized garlic derived SEVs by nanoparticle tracking analysis, electron microscopy and SEV surface antibodies. In order to investigate anti-cancer property of garlic SEVs A498 human kidney carcinoma, A549 human lung carcinoma were used as cell models along with the normal human dermal fibroblast cell lines. Annexin V/pI staining and analysis of apoptotic mRNA and protein expression levels suggested that garlic SEVs induced apoptosis through activation of intrinsic pathway. Furthermore, angiogenic VEGF protein expression levels significantly decreased in response to SEVs treatment in cancer cells. Our results support that garlic derived SEVs could cause apoptotic cell death among cancer cells while normal cells remain unaffected with the treatment. This study revealed for the first time that plant SEVs possess anti-cancer affects by inducing caspase mediated apoptosis and provided a new alternative for cancer treatment.

A robust optimization approach for the breast cancer targeted design of PEtOx-b-PLA polymersomes.

The equipping of nanoparticles with the peptide moiety recognizing a particular receptor, enables cell or tissue-specific targeting, therefore the optimization of the targeted nanoparticles is a key factor in the formulation design process. In this paper, we report the optimization concept of Doxorubicin encapsulating PEtOx-b-PLA polymersome formulation equipped with Peptide18, which is a breast cancer recognizing tumor homing peptide, and the unveiling of the cell-specific delivery potential. The most dominant formulation parameters, which are the polymer to Doxorubicin mass ratio (w/w) and the aqueous to organic phase ratio (v/v), were optimized using Central Composite Design (CCD) based Response Surface Methodology. The characteristics of optimum polymersome formulation were determined as the hydrodynamic diameter of 146.35 nm, the PDI value of 0.136, and the encapsulation efficiency of 57.11% and TEM imaging, which are in agreement with the DLS data, showed the spherical morphology of the polymersomes. In order to demonstrate the breast cancer-specific delivery of targeted polymersomes, the flow cytometry and confocal microscopy analyses were carried out. The targeted polymersomes were accumulated 8 times higher in AU565 cells compared to MCF10A cells and the intracellular Doxorubicin was almost 10 times higher in AU565 cells. The CCD-mediated optimized targeted polymersomes proposed in this report holds the promise of targeted therapy for breast cancer and can be potentially used for the development of novel treatments.

Synthesis and evaluation of tumor-homing peptides for targeting prostate cancer.

High toxicity caused by chemotherapeutic drugs and the acquisition of drug resistance by cancer cells are the major drawbacks in cancer therapy. A promising approach to overcome the posed barriers is conjugating tumor-homing peptides to drugs or nanocarriers. Such high-affinity peptides can specifically target surface markers overexpressed by cancer cells, ensuring a rapid and cancer-specific uptake of the drugs. Since prostate-specific membrane antigen (PSMA) is overexpressed by aggressive prostate cancer cells, targeting this surface protein with peptide conjugates can lead to the development of effective strategies against prostate cancer. In this study, we aimed to determine which PSMA-binding peptide among peptides 563, 562 and 9-mer, show the highest selectivity towards PSMA using 22Rv1 prostate cancer cells, a cell line with moderate PSMA levels. Tumor-homing peptides were synthesized by fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (Fmoc-SPPS) strategy, and evaluated for their prostate cancer cell-specific targeting efficiencies by flow cytometry. Our results showed that the PSMA-binding capacity of peptide 563 was superior to those of 562, 9-mer, and 5-mer; therefore, can be utilized as a potent-targeting agent not only in the treatment of high PSMA positive but also moderate PSMA positive prostate cancer tumors.

Design and evaluation of peptide-18-targeted nanoliposomes constructed by poly(2-oxazoline)-DOPE for doxorubicin delivery.

AIMS: The aim of this study is to develop targeted nanoliposome formulations to provide efficient treatment for breast cancer. In this study, peptide 18-modified poly(2-ethyl-2-oxazoline)-dioleoylphosphatidylethanolamine (P18-PEtOx-DOPE), was synthesised to construct nanoliposomes. METHODS: Doxorubicin (DOX) was encapsulated into the nanoliposomes by ethanol injection method. Particle size and polydispersity index were measured by dynamic light scattering. Zeta potential was determined by electrophoretic laser Doppler anemometry. The shape of the nanoliposomes was examined by transmission electron microscope. Specific bindings of P18-PEtOx-DOPE nanoliposomes were demonstrated on AU565 cells by confocal microscopy and flow cytometry studies. RESULTS: DOX-loaded nanoliposomes with particle diameter of 150.00 ± 2.84 nm and PDI of 0.212 ± 0.013 were obtained. PEtOx-DOPE and PEtOx-DOPE nanoliposomes are non-toxic on HUVEC, HEK293 and hMSC cells for 48 h. Furthermore, P18-PEtOx-DOPE nanoliposomes demonstrated specificity towards AU565 cells with high binding affinity. CONCLUSIONS: As a result, DOX-loaded P18-PEtOx-DOPE nanoliposomes can serve as favourable candidates in breast cancer targeted therapy.

Preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates.

COVID-19 outbreak caused by SARS-CoV-2 created an unprecedented health crisis since there is no vaccine for this novel virus. Therefore, SARS-CoV-2 vaccines have become crucial for reducing morbidity and mortality. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. The candidate vaccines in this study were OZG-3861 version 1 (V1), an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1), a GM-CSF adjuvant added vaccine. The candidate vaccines were applied intradermally to BALB/c mice to assess toxicity and immunogenicity. Preliminary results in vaccinated mice are reported in this study. Especially, the vaccine models containing GM-CSF caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature, when considered in terms of T and B cell responses. Another important finding was that the presence of adjuvant was more important in T cell in comparison with B cell response. Vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study shows that the vaccines are effective and leads us to start the challenge test to investigate the gamma-irradiated inactivated vaccine candidates for infective SARS-CoV-2 virus in humanized ACE2 + mice.

The Utilization of Poly(2-ethyl-2-oxazoline)-b-Poly(ε-caprolactone) Ellipsoidal Particles for Intracellular BIKDDA Delivery to Prostate Cancer.

Prostate cancer is the most common cancer, which is about 15-20% among male cancers worldwide. As most common strategies such as radiotherapy, chemotherapy, or surgery alone can be unsuccessful in the treatment of prostate cancer, this study aims to develop a new approach to deliver newly generated proapoptotic gene, BIKDDA, to androgen independent prostate cancer cells, 22RV1, using new generation nanocarriers called ellipsoids. As far as it is known, this is the first study that assesses the ability of proapoptotic gene BIKDDA to induce apoptosis in prostate cancer cell. BIKDDA encapsulating PEtOx-b-PCL-based ellipsoids are fabricated by solvent-switch method, and their morphology, size, and BIKDDA content are characterized. Gene delivery efficiency of BIKDDA loaded PEtOx-b-PCL ellipsoids is demonstrated by analysis of BIK mRNA expression with real-time PCR. The apoptotic effect of PEtOx-b-PCL ellipsoids loaded with BIKDDA (EPs-BIKDDA) on 22RV1 is shown by Annexin V staining. The obtained results demonstrate that the treatment of 22RV1 cells with EPs-BIKDDA can significantly increase BIK mRNA levels by 4.5-fold leading to cell death. This study not only represents BIKDDA as a potential therapeutic strategy in prostate cancer but also the capacity of ellipsoids as promising in vivo gene delivery vehicles.

Role of Tissue Transglutaminase Catalytic and Guanosine Triphosphate-Binding Domains in Renal Cell Carcinoma Progression.

Tissue transglutaminase (TG2) is a multifunctional protein that can act as a cross-linking enzyme, GTPase/ATPase, protein kinase, and protein disulfide isomerase. TG2 is involved in cell adhesion, migration, invasion, and growth, as well as epithelial-mesenchymal transition (EMT). Our previous findings indicate that the increased expression of TG2 in renal cell carcinoma (RCC) results in tumor metastasis with a significant decrease in disease- and cancer-specific survival outcome. Given the importance of the prometastatic activity of TG2 in RCC, in the present study, we aim to investigate the relative contribution of TG2's transamidase and guanosine triphosphate (GTP)-binding/GTPase activity in the cell migration, invasion, EMT, and cancer stemness of RCC. For this purpose, the mouse RCC cell line RenCa was transduced with wild-type-TG2 (wt-TG2), GTP-binding deficient-form TG2-R580A, transamidase-deficient form with low GTP-binding affinity TG2-C277S, and transamidase-inactive form TG2-W241A. Our results suggested that predominantly, GTP-binding activity of TG2 is responsible for cell migration and invasion. In addition, CD marker analysis and spheroid assay confirmed that GTP binding/GTPase activity of TG2 is important in the maintenance of mesenchymal character and the cancer stem cell profile. These findings support a prometastatic role for TG2 in RCC that is dependent on the GTP binding/GTPase activity of the enzyme.

Design of Fluorescent Probes for Bioorthogonal Labeling of Carbonylation in Live Cells.

With the rapid development of chemical biology, many diagnostic fluorophore-based tools were introduced to specific biomolecules by covalent binding. Bioorthogonal reactions have been widely utilized to manage challenges faced in clinical practice for early diagnosis and treatment of several tumor samples. Herein, we designed a small molecule fluorescent-based biosensor, 2Hydrazine-5nitrophenol (2Hzin5NP), which reacts with the carbonyl moiety of biomolecules through bioorthogonal reaction, therefore can be utilized for the detection of biomolecule carbonylation in various cancer cell lines. Our almost non-fluorescent chemical probe has a fast covalent binding with carbonyl moieties at neutral pH to form a stable fluorescent hydrazone product leading to a spectroscopic alteration in live cells. Microscopic and fluorometric analyses were used to distinguish the exogenous and endogenous ROS induced carbonylation profile in human dermal fibroblasts along with A498 primary site and ACHN metastatic site renal cell carcinoma (RRC) cell lines. Our results showed that carbonylation level that differs in response to exogenous and endogenous stress in healthy and cancer cells can be detected by the newly synthesized bioorthogonal fluorescent probe. Our results provide new insights into the development of novel bioorthogonal probes that can be utilized in site-specific carbonylation labeling to enhance new diagnostic approaches in cancer.

Association of 5'UTR polymorphism of secretory phospholipase A2 group IIA (PLA2G2A) gene with prostate cancer metastasis.

Phospholipase A2 (PLA2) enzymes are small lipolytic hydrolases that can regulate immune responses through generation of Arachidonic Acid (AA), a precursor molecule of lipid mediators like prostaglandins, leukotrienes and thromboxanes. One of the family members of PLA2, secretory Phospholipase A2 Group IIA (PLA2G2A), was associated with different types of malignancies including prostate cancer. Elevated serum levels of PLA2G2A was found in prostate cancer (PCa) patients and associated with increased tumor grade in literature. 5'UTR regions have regulatory role in protein expression by controlling the accessibility of factors necessary for the translation initiation. Single nucleotide polymorphisms at 5'UTR regions have the potential to affect mRNA translation efficiency resulting in altered protein levels depending on structure and nucleotide content. Given that the 5'UTR polymorphism in PLA2G2A gene (rs11573156) is associated with increased serum levels of PLA2G2A, the association of this 5'UTR polymorphism with PCa susceptibility and metastasis was investigated in this study. Total of 261 PCa patients and 128 control individuals were genotyped with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Individuals with heterozygous CG genotype was found to have significantly reduced risk of PCa metastasis with an Odds Ratio (OR) of 0.405 (p = 0.028, 95%CI = 0.181-0.906), compared to the carriers of homozygous CC genotype (p > 0.05) suggesting an anti-metastatic effect for the G allele. No association was found between PCa susceptibility and Gleason score (p > 0.05) in Turkish population.

Tissue transglutaminase_variant 2-transduced mesenchymal stem cells and their chondrogenic potential.

As cartilage is incapable of self-healing upon severe degeneration because of the lack of blood vessels, cartilage tissue engineering is gaining importance in the treatment of cartilage defects. This study was designed to improve cartilage tissue regeneration by expressing tissue transglutaminase variant 2 (TGM2_v2) in mesenchymal stem cells (MSC) derived from bone marrow of rats. For this purpose, rat MSCs transduced with TGM2_v2 were grown and differentiated on three-dimensional polybutylene succinate (PBSu) and poly-l-lactide (PLLA) blend scaffolds. The transduced cells could not only successfully express the short form transglutaminase-2, but also deposited the protein onto the scaffolds. In addition, they could spontaneously produce cartilage-specific proteins without any chondrogenic induction, suggesting that TGM2_v2 expression provided the cells the ability of chondrogenic differentiation. PBSu:PLLA scaffolds loaded with TGM2_v2 expressing MSCs could be used in repair of articular cartilage defects.

Palbociclib, a selective CDK4/6 inhibitor, restricts cell survival and epithelial-mesenchymal transition in Panc-1 and MiaPaCa-2 pancreatic cancer cells.

The mortality rate of pancreatic cancer has close parallels to its incidence rate because of limited therapeutics and lack of effective prognosis. Despite various novel chemotherapeutics combinations, the 5-year survival rate is still under 5%. In the current study, we aimed to modulate the aberrantly activated PI3K/AKT pathway and epithelial-mesenchymal transition (EMT) signaling with the treatment of CDK4/6 inhibitor PD-0332991 (palbociclib) in Panc-1 and MiaPaCa-2 pancreatic cancer cells. It was found that PD-0332991 effectively reduced cell viability and proliferation dose-dependently within 24 hours. In addition, PD-0332991 induced cell cycle arrest at the G1 phase by downregulation of aberrant expression of CDK4/6 through the dephosphorylation of Rb in each cell lines. Although PD-0332991 treatment increased epithelial markers and decreased mesenchymal markers, the nuclear translocation of ß-catenin was not prevented by PD-0332991 treatment, especially in MiaPaCa-2 cells. Effects of PD-0332991 on the regulation of PI3K/AKT signaling and its downstream targets such as GSK-3 were cell type-dependent. Although the activity of AKT was inhibited in both cell lines, the phosphorylation of GSK-3ß at Ser9 increased only in Panc-1. In conclusion, PD-0332991 induced cell cycle arrest and reduced the cell viability of Panc-1 and MiaPaCa-2 cells. However, PD-0332991 differentially affects the regulation of the PI3K/AKT pathway and EMT process in cells due to its distinct influence on Rb and GSK-3/ß-catenin signaling. Understanding the effect of PD-0332991 on the aberrantly activated signaling axis may put forward a new therapeutic strategy to reduce the cell viability and metastatic process of pancreatic cancer.

Hypericum olympicum L. recovers DNA damage and prevents MMP-9 activation induced by UVB in human dermal fibroblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum olympicum L. (Hypericaceae) flowering aerial parts has been utilized in Turkish folk medicine as a remedy against inflamed skin problems. AIM OF THE STUDY: This study was designed to state the effect of H. olympicum on dermatological problems. For this purpose effect of the plant extract on the DNA strand break and matrix metalloproteinase (MMP)-9 activity of human dermal fibroblast (HDFs) cells irradiated with UVB as well as antioxidant activity potential were studied. MATERIALS AND METHODS: The methanolic extract of Hypericum olympicum (HOM) was prepared by maceration at room temperature. DNA damage and increased MMP-9 activity in HDFs were induced by UVB irradiation. The cell viability was measured by water-soluble tetrazolium salt (WST)-1 assay. The effects on DNA strand break was investigated by single gel electrophoresis (commonly known as Comet assay), while MMP-9 activity was observed by gelatin zymography assay. In vitro antioxidant tests were performed to indicate the effect on reactive oxygen species (ROS). The major metabolites were identified and their concentrations were measured by high performance thin layer chromatography (HPTLC). RESULTS: HOM was found to recover DNA damage dose-dependently. The enzymatic activity of MMP-9 was inhibited almost 100% by the treatment of 1.5 mg/mL of the extract. It also enhanced cell proliferation in those cells, and also it was shown to possess antioxidant activity. The major metabolites of HOM were identified as chlorogenic acid and quercetin glycosides (rutin, hyperoside, isoquercitrin). CONCLUSION: Experimental studies have proven the traditional use of Hypericum olympicum in inflamed skin problems acting by inhibition of the inflammatory pathway and recovery of DNA damage induced experimentally.

Dual-Inhibition of mTOR and Bcl-2 Enhances the Anti-tumor Effect of Everolimus against Renal Cell Carcinoma In Vitro and In Vivo.

Renal cell carcinoma (RCC) is the predominant type of kidney cancer. Mammalian target of rapamycin (mTOR) inhibitor everolimus is currently used as a second-line therapy for sorafenib or sunitinib-refractory metastatic RCC patients. The clinical limitation confronted during everolimus therapy is the onset of drug resistance that decreases the efficacy of the drug. Elevated level of anti-apoptotic Bcl-2 protein is proposed to be an emerging feedback loop for the acquired drug-resistance in various cancer types. In this study, the Bcl-2 inhibitor ABT-737 was used in combination with everolimus to enhance its anti-tumor effectiveness in everolimus-resistant RCC cell lines. Everolimus and ABT-737 combination synergistically led to a decrease in the proliferation of primary site A-498 and metastatic site Caki-1 RCC cell lines, which was accompanied by a reduction in protein levels of cell cycle and mTOR pathway proteins. In both RCC cell lines, everolimus-ABT-737 combination not only induced apoptosis, caspase and PARP-1 cleavage but also a decrease in Bcl-2 protein levels in parallel with a concomitant increase in Bim and Noxa levels. In order to confirm our in vitro findings, we have generated everolimus-resistant RenCa cell line (RenCares) to establish a RCC mouse xenograft model. Animals co-treated with everolimus and ABT-737 exhibited a complete suppression of tumor growth without any notable toxicity. This study thus proposes the everolimus-ABT-737 combination as a novel therapeutic strategy for the treatment of RCC to overcome the current clinical problem of everolimus resistance.