Generation of anti-SN38 antibody for loading efficacy and therapeutic monitoring of SN38-containing therapeutics.

SN38, one of the most potent anti-tumor analogues of the camptothecins (CPTs), has limitations in its direct formulation as an anticancer agent due to its super toxicity and poor solubility in water and pharmaceutically approved solvents. However, it has garnered significant scientific interest as a payload in conjugated nanomedicine platforms (e.g., SN-38lip, NK012, SNB-101, and ADCs) to enhance their effectiveness and safety. The development of these platforms necessitates a convenient quantitative determination of SN38 in preclinical and clinical studies, a need that our study directly addresses, offering a practical solution to a pressing problem in cancer research and drug development. This study details the meticulous process of generating poly and monoclonal antibodies (pAb and mAb) against SN38 and their application to measure the SN38 in naked and conjugated forms of SN38-conjugated ADCs. For this purpose, two haptens of SN38 were synthesized by introducing the glycine or 4-amino-4-oxobutanyol(glycine) moiety as a conjugation functional group of the SN38. IR, NMR and mass spectrometric techniques confirmed the chemical modifications of the haptens. The haptens were then conjugated to each bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) protein. The SN38-KLH conjugates were meticulously examined for immunization and generation of pAb and mAb. The immunization efficiency, reactivity, binding affinity, specificity, and cross-reactivity of purified pAb and mAb against Irinotecan, a model for the emergence of an SN38 derivative in clinical settings, were evaluated using ELISA and western blotting (WB) techniques. Conjugation efficiency of the SN38 to the KLH was increased using 4-amino-4-oxobutanyol(glycine) moiety, as its immunization efficacy was more to generate pAb. Furthermore, only this hapten could immunized mice to generate mAb recognizing SN38 with nanomolar equilibrium affinity. Our recent findings strongly support the notion that the generated pAb employed in developing an ELISA effectively ascertains the presence of SN38 in SN38-conjugated ADC, with a test midpoint EC50 of 2.5 µg/mL. Our study's unique contribution to the field lies in the development of specific antibodies against SN38 for measuring it on ADC, a feat that has not been achieved before. These immunoassays can be readily applied to detect other SN38-conjugate therapeutic platforms, thereby enhancing their clinical knowledge translation. The affinity of both pAb and mAb also meets the acceptance criteria for quantifying SN38 in fluidic material, as well as in Therapeutic drug monitoring (TDM) studies, a crucial aspect of personalized medicine. The potential applications of the anti-SN38 antibodies extend to reducing SN38-induced systemic toxicity through an inverse targeting strategy, a novel approach that piques further interest in our findings.

Inhibition of PI3K/AKT signaling using BKM120 reduced the proliferation and migration potentials of colorectal cancer cells and enhanced cisplatin-induced cytotoxicity.

BACKGROUND: Although extensive efforts have been made to improve the treatment of colorectal cancer (CRC) patients, the prognosis for these patients remains poor. A wide range of anti-cancer agents has been applied to ameliorate the clinical management of CRC patients; however, drug resistance develops in nearly all patients. Based on the prominent role of PI3K/AKT signaling in the development of CRC and current interest in the application of PI3K inhibitors, we aimed to disclose the exact mechanism underlying the efficacy of BKM120, a well-known pan-class I PI3K inhibitor, in CRC-derived SW480 cells. MATERIALS AND METHODS: The effects of BKM120 on SW480 cells were studied using MTT assay, cell cycle assay, Annexin V/PI apoptosis tests, and scratch assay. In the next step, qRT-PCR was used to investigate the underlying molecular mechanisms by which the PI3K inhibitor could suppress the survival of SW480 cells. RESULT: The results of the MTT assay showed that BKM120 could decrease the metabolic activity of SW480 cells in a concentration and time-dependent manner. Investigating the exact mechanism of BKM120 showed that this PI3K inhibitor induces its anti-survival effects through a G2/M cell cycle arrest and apoptosis-mediated cell death. Moreover, the scratch assay demonstrated that PI3K inhibition led to the inhibition of cancer invasion and inhibition of PI3K/AKT signaling remarkably sensitized SW480 cells to Cisplatin. CONCLUSION: Based on our results, inhibition of PI3K/AKT signaling can be a promising approach, either as a single modality or in combination with Cisplatin. However, further clinical studies should be performed to improve our understanding.

In vitro and in silico characterization of a novel glutamate carboxypeptidase from Cohnella sp. A01.

Glutamate carboxypeptidase is a bacterial enzyme of metallopeptidase superfamily. This enzyme is an exo-peptidase that catalyzes the hydrolysis of glutamate residues at the C-terminus of folic acid. The rCP302 is a novel zinc ion-dependent recombinant glutamate carboxypeptidase derived from a thermophilic bacterium, Cohnella sp. A01 (PTCC No: 1921). By simulating the structure of rCP302, analyzing its activity in various environmental settings, and contrasting it with that of related enzymes, we wanted to evaluate the heterologous production, purification, and characterization of this enzyme. The bioinformatics study showed that rCP302 had maximum similarity to M20 family of metallopeptidases. The purified rCP302 molecular weight was about 41.6 kDa. The optimum temperature and pH for the catalytic activity of rCP302 were 50 °C and 7.2, respectively. Fluorescence spectroscopy data elucidated the secondary structure of rCP302 and determined conformational changes caused by alterations in ambient conditions. Using folate as a substrate, Km and specific activity values were calculated as 0.108 µM and 687 µmol/min/mg, respectively. The enzyme activity was strongly inhibited when EDTA sequestered zinc ions. The half-life of this enzyme at 30 °C was 2012 min. Regarding the ability of rCP302 to degrade folic acid, and its long half-life at 37 °C, the normal temperature of many mammals, this enzyme can be introduced for further study for use in the pharmaceutical industry.

Nano-bio interaction: An overview on the biochemical binding of DNA to inorganic nanoparticles for the development of anticancer and antibacterial nano-platforms.

It has long been known that inorganic nanoparticles (NPs) can interact with biological macromolecules and show a wider range of biomedical characteristics, including antibacterial, anticancer and antioxidant effects, which cannot be mimicked by their bulky counterparts. It is of great importance in their biomedical applications to study DNA damage in bacterial and cancer cells to develop biocompatible therapeutic nano-platforms derived from inorganic NPs. Therefore, to determine how DNA interacts with inorganic NPs serving as therapeutic agents, thermodynamic and structural studies are essential for an understanding of those mechanisms, thereby allowing for their modulation and manipulation of nano-bio interface. In this paper, we aimed to overview the biophysical techniques typically employ to study DNA-NP interactions as well as the mechanistic aspects of the interaction between different inorganic NPs and calf thymus DNA (CT-DNA), a well-known laboratory model, followed by a survey of different parameters affecting the interaction of NPs and DNA. The molecular interactions between inorganic NPs and DNA were then discussed in relation to their anticancer and antibacterial properties. As a final point, we discussed challenges and future perspectives to put forward the possible applications of the field. In conclusion, the interaction between NPs and DNA needs to be studied more deeply in order to develop potential NP-based anticancer and antibacterial platforms for future clinical applications.

BK polyomavirus in Iranian patients with papillary thyroid cancer: Is it a major future challenge?

BK polyomavirus (BKPyV) is a well-known cause of nephropathy in renal transplant recipients. It has recently received much attention from researchers as a major predisposing factor for various cancers. This study aimed to investigate how BKPyV affected the advancement of papillary thyroid carcinoma (PTC). A total of 1057 samples were tested for BKPyV DNA and RNA, comprising 645 paraffin-embedded PTC biopsy samples (PEBS), 412 fresh biopsy samples (FBS), and 1057 adjacent noncancerous samples. The BKPyV DNA was found in 511 (48.3%) of the specimens, including 347 (84.2%) FBS and 164 (25.4%) PEBS. The mean BKPyV copy number was significantly lower in patients with PEBS (0.5 × 10-4 ± 0.1 × 10-4 copies/cell) than in FBS (1.3 × 10-1 ± 0.2 × 10-1 copies/cell) and non-PTC normal samples (0.3 × 10-5 ± 0.04 × 10-5 copies/cell). The PEBS had lower LT-Ag RNA expression than FBS, and no VP1 gene transcript expression was detected. In conclusion, although our findings indicated the presence of BKPyV in some Iranian PTC patients, more research is needed to corroborate these findings.

An Updated Review on EPR-Based Solid Tumor Targeting Nanocarriers for Cancer Treatment.

The enhanced permeability and retention (EPR) effect in cancer treatment is one of the key mechanisms that enables drug accumulation at the tumor site. However, despite a plethora of virus/inorganic/organic-based nanocarriers designed to rely on the EPR effect to effectively target tumors, most have failed in the clinic. It seems that the non-compliance of research activities with clinical trials, goals unrelated to the EPR effect, and lack of awareness of the impact of solid tumor structure and interactions on the performance of drug nanocarriers have intensified this dissatisfaction. As such, the asymmetric growth and structural complexity of solid tumors, physicochemical properties of drug nanocarriers, EPR analytical combination tools, and EPR description goals should be considered to improve EPR-based cancer therapeutics. This review provides valuable insights into the limitations of the EPR effect in therapeutic efficacy and reports crucial perspectives on how the EPR effect can be modulated to improve the therapeutic effects of nanomedicine.

Loading Pistacia atlantica essential oil in solid lipid nanoparticles and its effect on apoptosis of breast cancer cell line MDA-MB-231.

Pistacia atlantica has an anti-cancer effect due to its essential oil which is the major constituent of P. atlantica. Unfortunately, this essential oil evaporates easily and makes it less effective. The current research, therefore, aimed to improve the anti-cancer effect of P. atlantica essential oil (PAEO) in solid lipid nanoparticles (SLN). The chemical components of PAEO were assessed by gas chromatography. PAEO-SLNs were prepared by the probe-ultrasonication method, and their particle size, polydispersity index and zeta potential were determined. Encapsulation Efficiency (EE%) and Loading Capacity (LC%) of formulations was also calculated. Transmission electron microscopy was employed to determine the morphology of optimal formulation (PAEO-SLN4). Furthermore, the anticancer effects of PAEO-SLN4 against MDA-MB-231 cells were evaluated by cellular assays. The results showed that the type of surfactant and loading of the essential oil had a significant effect on size distribution, zeta potential and the polydispersity index. The encapsulation efficiency (EE%) and loading capacity for PAEO-SLN4 were 97.3% and 9.6%, respectively. The cellular assay demonstrates that PAEO-SLN4 could lead MDA-MB-231 cells to apoptosis. The findings also revealed that PAEO-SLN4 can stimulate apoptosis in MDA-MB-231 cells more than the placebo and free PAEO thereby indicating PAEO-SLN4 to be beneficial in breast cancer treatment.

Exploring the Interaction of Cobalt Oxide Nanoparticles with Albumin, Leukemia Cancer Cells and Pathogenic Bacteria by Multispectroscopic, Docking, Cellular and Antibacterial Approaches.

AIM: The interaction of NPs with biological systems may reveal useful details about their pharmacodynamic, anticancer and antibacterial effects. METHODS: Herein, the interaction of as-synthesized Co3O4 NPs with HSA was explored by different kinds of fluorescence and CD spectroscopic methods, as well as molecular docking studies. Also, the anticancer effect of Co3O4 NPs against leukemia K562 cells was investigated by MTT, LDH, caspase, real-time PCR, ROS, cell cycle, and apoptosis assays. Afterwards, the antibacterial effects of Co3O4 NPs against three pathogenic bacteria were disclosed by antibacterial assays. RESULTS: Different characterization methods such as TEM, DLS, zeta potential and XRD studies proved that fabricated Co3O4 NPs by sol-gel method have a diameter of around 50 nm, hydrodynamic radius of 177 nm with a charge distribution of -33.04 mV and a well-defined crystalline phase. Intrinsic, extrinsic, and synchronous fluorescence as well as CD studies, respectively, showed that the HSA undergoes some fluorescence quenching, minor conformational changes, microenvironmental changes as well as no structural changes in the secondary structure, after interaction with Co3O4 NPs. Molecular docking results also verified that the spherical clusters with a dimension of 1.5 nm exhibit the most binding energy with HSA molecules. Anticancer assays demonstrated that Co3O4 NPs can selectively lead to the reduction of K562 cell viability through the cell membrane damage, activation of caspase-9, -8 and -3, elevation of Bax/Bcl-2 mRNA ratio, ROS production, cell cycle arrest, and apoptosis. Finally, antibacterial assays disclosed that Co3O4 NPs can stimulate a promising antibacterial effect against pathogenic bacteria. CONCLUSION: In general, these observations can provide useful information for the early stages of nanomaterial applications in therapeutic platforms.

Expression of Diverse Angiogenesis Factor in Different Stages of the 4T1 Tumor as a Mouse Model of Triple-Negative Breast Cancer.

Purpose: Triple-negative breast cancer (TNBC) is specified by high vascularity and repetitious metastasis. Although several studies have indicated that angiogenesis has an important role in invasive breast cancer, a suitable model of TNBC that can show the exact onset of angiogenesis factors still needs to be developed. The purpose of this study is to determine the expression level of angiogenesis factors in different clinical stages of the 4T1 tumor as TNBC mouse model. Methods: Twenty mice were injected by the 4T1 cell line, and four mice selected as healthy controls. Following by tumor induction, the mice were randomly put into four groups, each contains four mice. Once the tumor volume reached to the early stage (<100 mm3), intermediate stage (100-300 mm3), advanced stage (300-500 mm3), and end stage (>500 mm3), they were removed by surgery. Then, the expression levels of Hif1α, VEGFR1, and VEGFR2 genes, as well as tumor markers of VEGF, bFGF and CD31, were evaluated by qPCR and immunohistochemistry (IHC) respectively. The statistical analysis was done by SPSS version 16. Results: TNBC tumors were confirmed and multi-foci metastasis in the lung were seen. The mRNA and protein expression levels of the angiogenesis factors increased in the early stage and as the tumor grew, their expression level enhanced dramatically. Conclusion: The 4T1 syngeneic mouse tumor may serve as an appropriate TNBC model for further investigation of the angiogenesis and therapies. Moreover, angiogenesis factors are induced before the advanced stage, and anti-angiogenesis therapy is necessary to be considered at the first line of treatment in TBNC.

Vitamin K1 As A Potential Molecule For Reducing Single-Walled Carbon Nanotubes-Stimulated α-Synuclein Structural Changes And Cytotoxicity.

AIMS: Different kinds of vitamins can be used as promising candidates to mitigate the structural changes of proteins and associated cytotoxicity stimulated by NPs. Therefore, the structural changes of α-syn molecules and their associated cytotoxicity in the presence of SWCNTs either alone or co-incubated with vitamin K1 were studied by spectroscopic, bioinformatical, and cellular assays. METHODS: Intrinsic and ThT fluorescence, CD, and Congo red absorption spectroscopic approaches as well as TEM investigation, molecular docking, and molecular dynamics were used to explore the protective effect of vitamin K1 on the structural changes of α-syn induced by SWCNTs. The cytotoxicity of α-syn/SWCNTs co-incubated with vitamin K1 against SH-SY5Y cells was also carried out by MTT, LDH, and caspase-3 assays. RESULTS: Fluorescence spectroscopy showed that vitamin K1 has a significant effect in reducing SWCNT-induced fluorescence quenching and aggregation of α- syn. CD, Congo red adsorption, and TEM investigations determined that co-incubation of α- syn with vitamin K1 inhibited the propensity of α-syn into the structural changes and amorphous aggregation in the presence of SWCNT. Docking studies determined the occupation of preferred docked site of SWCNT by vitamin K1 on α- syn conformation. A molecular dynamics study also showed that vitamin K1 reduced the structural changes of α- syn induced by SWCNT. Cellular data exhibited that the cytotoxicity of α- syn co-incubated with vitamin K1 in the presence of SWCNTs is less than the outcomes obtained in the absence of the vitamin K1. CONCLUSION: It may be concluded that vitamin K1 decreases the propensity of α- syn aggregation in the presence of SWCNTs and induction of cytotoxicity.

Inhibition of PI3K pathway using BKM120 intensified the chemo-sensitivity of breast cancer cells to arsenic trioxide (ATO).

Although conventional therapeutic approaches have brought remarkable advantages for the treatment of breast cancer (BC), drug-resistance still remains as a leading cause of tumor recurrence in this malignancy. In the present study, we designed experiments to evaluate the therapeutic value of PI3K inhibition in combination with Arsenic trioxide (ATO) in MCF-7 cells. The results of our study manifested that BKM120 sensitized MCF-7 cells to the lower concentrations of ATO. The significant anti-cancer effect of PI3K inhibition became even more evident when we found that BKM120, either as a single agent or in combination with ATO, reduced clonogenic ability of anoikis-resistant stem-like MCF-7 cells. Our findings also showed that BKM120 augmented ATO-induced anti-proliferative effects through inducing G1 arrest and reducing the incorporation of BrdU into the synthesized DNA of drugs-treated cells, which was coupled with c-Myc-mediated suppression of hTERT expression. Moreover, we found that in the presence of PI3K inhibitor, ATO is able to more profoundly induce apoptosis in MCF-7 cells, as revealed by the increment in the percentage of haplodiploid sub-G1 cells and the externalization of phosphatidylserine. Real-time PCR analysis also revealed that probably down-regulation of survivin coupled with up-regulation of forkhead family transcription factors is responsible for the enhancive effect of drugs in this cell line. Conclusively, this study shed lights on the effect of PI3K inhibition in chemo-sensitivity of MCF-7 cells, disclosing that combination of BKM120 and ATO could be a promising therapeutic approach in BC.

Albumin binding, anticancer and antibacterial properties of synthesized zero valent iron nanoparticles.

BACKGROUND: Nanoparticles (NPs) have been emerging as potential players in modern medicine with clinical applications ranging from therapeutic purposes to antimicrobial agents. However, before applications in medical agents, some in vitro studies should be done to explore their biological responses. AIM: In this study, protein binding, anticancer and antibacterial activates of zero valent iron (ZVFe) were explored. MATERIALS AND METHODS: ZVFe nanoparticles were synthesized and fully characterized by X-ray diffraction, field-emission scanning electron microscope, and dynamic light scattering analyses. Afterward, the interaction of ZVFe NPs with human serum albumin (HSA) was examined using a range of techniques including intrinsic fluorescence, circular dichroism, and UV-visible spectroscopic methods. Molecular docking study was run to determine the kind of interaction between ZVFe NPs and HSA. The anticancer influence of ZVFe NPs on SH-SY5Y was examined by MTT and flow cytometry analysis, whereas human white blood cells were used as the control cell. Also, the antibacterial effect of ZVFe NPs was examined on Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), and Staphylococcus aureus (ATCC 25923). RESULTS: X-ray diffraction, transmission electron microscope, and dynamic light scattering analyses verified the synthesis of ZVFe NPs in a nanosized diameter. Fluorescence spectroscopy analysis showed that ZVFe NPs spontaneously formed a complex with HSA through hydrogen bonds and van der Waals interactions. Also, circular dichroism spectroscopy study revealed that ZVFe NPs did not change the secondary structure of HSA. Moreover, UV-visible data presented that melting temperature (Tm) of HSA in the absence and presence of ZVFe NPs was almost identical. Molecular dynamic study also showed that ZVFe NP came into contact with polar residues on the surface of HSA molecule. Cellular assays showed that ZVFe NPs can induce cell mortality in a dose-dependent manner against SH-SY5Y cells, whereas these NPs did not trigger significant cell mortality against normal white bloods in the concentration range studied (1-100 µg/mL). Antibacterial assays showed a noteworthy inhibition on both bacterial strains. CONCLUSION: In conclusion, it was revealed that ZVFe NPs did not induce a substantial influence on the structure of protein and cytotoxicity against normal cell, whereas they derived significant anticancer and antibacterial effects.

Biophysical, bioinformatical, cellular, and molecular investigations on the effects of graphene oxide nanosheets on the hemoglobin structure and lymphocyte cell cytotoxicity.

BACKGROUND: Implementations of nanoparticles have been receiving great interest in medicine and technology due to their unique characteristics. However, their toxic impacts on the biological system are not well explored. AIM: This study aims to investigate the influence of fabricated nano graphene oxide (NGO) sheets on the secondary and quaternary structural alterations of human hemoglobin (Hb) and cytotoxicity against lymphocyte cells. MATERIALS AND METHODS: Different spectroscopic methods, such as extrinsic and synchronous fluorescence spectroscopy and far circular dichroism (CD) spectroscopy, molecular docking investigation, cellular assays (trypan blue exclusion, cellular uptake, ROS, cell cycle, and apoptosis), and molecular assay (fold changes in anti/proapoptotic genes [B-cell lymphoma-2 {BCL2}/BAX] expression levels) were used in this study. RESULTS: Transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and zeta potential investigations revealed the nano-sized nature of NGOs with good colloidal stability. Extrinsic fluorescence spectroscopy by using 8-anilinonaphthalene-1 -sulfonic acid and synchronous fluorescence spectroscopy showed that NGOs can unfold the quaternary structure of Hb in the vicinity of Tyr residues. The CD investigation demonstrated that the α-helicity of Hb experienced substantial alteration upon interaction with increasing concentrations of NGOs. The molecular docking study showed that NGOs interacted with polar residues of Hb. Cellular and molecular assays revealed that NGOs lead to ROS formation, cell cycle arrest, and apoptosis through the BAX and BCL2 pathway. CONCLUSION: These data reveal that NGOs can induce some protein structural changes and stimulate cytotoxicity against normal cell targets. Therefore, their applications in healthy systems should be limited.

Interaction of silica nanoparticles with tau proteins and PC12 cells: Colloidal stability, thermodynamic, docking, and cellular studies.

Study on the side effects of the nanoparticles (NPs) can provide useful information regarding their biological and medical applications. Herein, the colloidal stability of the silicon dioxide NPs (SiO2 NPs) in the absence and presence of tau was investigated by TEM and DLS techniques. Afterwards, the thermodynamic parameters of interaction between SiO2 NPs and tau were determined by fluorescence spectroscopy and docking studies. Finally, the cytotoxic effects of SiO2 NPs on the viability of PC12 cells were investigated by MTT, AO/EB staining and flow cytometry assays. TEM, DLS, and zeta potential investigations revealed that tau can reduce the colloidal stability of SiO2 NPs. Fluorescence spectroscopy study indicated that SiO2 NPs bound to the tau with high affinity through hydrogen bonds and van der Waals interactions. Docking study also determined that Ser, Thr and Tyr residues provide a polar microenvironment for SiO2 NPs/tau interaction. Cellular studies demonstrated that SiO2 NPs can induce cell mortality through both apoptosis and necrosis mechanisms. Therefore, it may be concluded that the biological systems such as nervous system proteins can affect the colloidal stability of NPs and vice versa NPs in the biological systems can bind to proteins and cell membranes non-specifically and may induce toxicity.

The Effect of Pentoxifylline on bcl-2 Gene Expression Changes in Hippocampus after Ischemia-Reperfusion in Wistar Rats by a Quatitative RT-PCR Method.

Ischemia-reperfusion injury is the tissue damage caused when blood supply returns to the tissue after a period of ischemia or lack of oxygen. Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines. Twenty-four male Wistar rats (250-300 g body wt) were used in this study. The animals were divided into four groups of 6 rats each: I: Control group that was subjected to ischemia-reperfusion, II: Ischemia-reperfusion group that was subjected to all surgical procedures, III: Drug group that received pentoxifylline (200, 400 and 600 mg/kg) 60 min before and after ischemia and IV: Vehicle group that received saline. Seventy two h after ischemia-reperfusion, the hippocampus was taken for studying the changes in bcl-2 gene expression. We used quantitative real-time PCR for the detection of bcl-2 gene expression in ischemia and drug groups and then compared them to normal samples. The results showed the gene dosage ratio of 0.66 and 1.5 for ischemia group and the drug groups, respectively. The results also showed the bcl-2 gene expression declined in ischemia group as compared to the drug group. Furthermore, we observed a significant difference in the bcl-2 gene expression between ischemia and drug groups. These findings are consistent with anti-apoptotic properties of bcl-2 gene. Furthermore this method provides a powerful tool for the investigators to study brain ischemia and respond to the treatment drugs with anti-apoptotic agents.