Synchronized Codelivery of Combination Chemotherapies Intratumorally Using a Lipidic Lyotropic Liquid Crystal System.

In this work, an injectable in situ depot-forming lipidic lyotropic liquid crystal (L3C) system is developed to codeliver a precisely synchronized combination of chemotherapeutics intratumorally. The developed L3C system is composed of amphiphilic lipids and surfactants, including monoolein, phosphatidylcholine, tocopherol acetate, and d-α-tocopherol polyethylene glycol 1000 succinate. Owing to its amphiphilic nature, the developed formulation can coaccommodate both hydrophobic and hydrophilic chemotherapeutic moieties simultaneously. The study presents a proof of concept by designing a combination chemotherapy regimen in vitro and demonstrating its in vivo translation using doxorubicin and paclitaxel as model hydrophilic and hydrophobic drug moieties, respectively. The synchronized combination of the two chemotherapeutics with maximum synergistic activity was identified, coloaded in the developed L3C system at predefined stoichiometric ratios, and evaluated for antitumor efficacy in the 4T1 breast tumor model in BALB/c mice. The drug-loaded L3C formulation is a low-viscosity injectable fluid with a lamellar phase that transforms into a hexagonal mesophase depot system upon intratumoral injection. The drug-loaded depot system locally provides sustained intratumoral delivery of the chemotherapeutics combination at their precisely synchronized ratio for over a period of one month. Results demonstrate that the exposure of the tumor to the precisely synchronized intratumoral chemotherapeutics combination via the developed L3C system resulted in significantly higher antitumor activity and reduced cardiotoxicity compared to the unsynchronized combination chemotherapy or the synchronized but uncoordinated drug delivery administered by a conventional intravenous route. These findings demonstrate the potential of the developed L3C system for achieving synchronized codelivery of the chemotherapeutics combination intratumorally and improving the efficacy of combination chemotherapy.

An Injectable In Situ Depot-Forming Lipidic Lyotropic Liquid Crystal System for Localized Intratumoral Drug Delivery.

To address the need for localized chemotherapy against unresectable solid tumors, an injectable in situ depot-forming lipidic lyotropic liquid crystal system (L3CS) is explored that can provide spatiotemporal control over drug delivery. Although liquid crystals have been studied extensively before but their application as an injectable intratumoral depot system for locoregional chemotherapy has not been explored yet. The developed L3CS in the present study is a low-viscosity injectable fluid having a lamellar phase, which transforms into a hexagonal mesophase depot system on subcutaneous or intratumoral injection. The transformed depot system can be preprogrammed to provide tailored drug release intratumorally, over a period of one week to one month. To establish the efficacy of the developed L3CS, doxorubicin is used as a model drug. The drug release mechanism is studied in detail both in vitro and in vivo, and the efficacy of the developed system is investigated in the murine 4T1 tumor model. The direct intratumoral injection of the L3CS provided localized delivery of doxorubicin inside the tumor and restricted its access within the tumor only for a sustained period of time. This led to an over 10-fold reduction in tumor burden, reduced cardiotoxicity, and a significant increase in the median survival rate, compared to the control group. The developed L3CS thus provides an efficient strategy for localized chemotherapy against unresectable solid tumors with a great degree of spatial and temporal control over drug delivery.

Efficient hydrolytic cleavage of DNA and antiproliferative effect on human cancer cells by two dinuclear Cu(II) complexes containing a carbohydrazone ligand and 1,10-phenanthroline as a coligand.

We report the synthesis, crystal structures and biological activities of two dinuclear Cu(II) complexes [Cu(o-phen)LCu(OAc)] (1) and [Cu(o-phen)LCu(o-phen)](OAc) (2), where o-phen = 1,10-phenanthroline, H3L = o-HOC6H4C(H)=N-NH-C(OH)=N-N=C(H)-C6H4OH-o, and OAc=CH3COO-. Both compounds display strong and broad X-band EPR spectra at RT in their powder state confirming that these are paramagnetic. The intercalative DNA binding of the compounds as revealed from spectrophotometric studies was found to be consistent with the results of fluorescence spectroscopic studies for ethidium bromide displacement assay as well as enhanced viscosity of DNA in the presence of these compounds. The compounds effectively catalyze hydrolytic cleavage of supercoiled pUC19 DNA and show remarkable cytotoxicity toward human lung cancer A549 cell line (IC50 values are 4.34 and 8.46 µM for 1 and 2, respectively) and breast cancer MCF7 cell line (IC50 values are 6.50 and 8.68 µM for 1 and 2, respectively) and are found to be relatively less toxic toward keratinocyte HaCaT normal cell line (IC50 values are 11.19 and 16.01 µM for 1 and 2, respectively). Annexin-V/PI dual staining results analyzed by flow cytometry strongly suggest the induction of apoptotic pathway for the anticancer activity of these complexes. Flow cytometry experiment for cell cycle analysis showed considerable increase in the G2/M phase in both A549 and MCF7 cell lines by these two compounds. On the other hand, compounds 1 and 2 activate reactive oxygen species (ROS) level in A549 cells, but act as scavengers or inhibitors of ROS in MCF7 cell line as analyzed by DCFDA staining using flow cytometry. Two dinuclear Cu(II) complexes exhibit efficient hydrolytic cleavage of DNA and display remarkable cytotoxicity against human lung cancer A549 and breast cancer MCF7cells. The ROS level in A549 cells is activated, but the ROS level in MCF7 cells is decreased in the presence of these complexes. Cell cycle analysis by flow cytometry shows G2/M phase arrest in both these cell lines.

DNA binding, cleavage and cytotoxicity studies of three mononuclear Cu(II) chloro-complexes containing N-S donor Schiff base ligands.

We report the biological activity of three Cu(II) complexes [Cu(pabt)Cl] (1), [Cu(pma)Cl] (2), and [Cu(pdta)Cl]Cl (3) (pabt = N-(2-mercaptophenyl)-2'-pyridylmethylenimine, pma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta = 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide). 1-3 display four-line EPR multiplet in solution at RT suggesting that these are mononuclear. DNA-binding studies using spectrophotometric titration of these complexes with calf thymus DNA showed binding through intercalation mode which was found to be consistent with the observation of increased viscosity of DNA and quenching of fluorescence of ethidium bromide bound DNA in the presence of these complexes. All three complexes were found to be efficient in bringing about oxidative and hydrolytic cleavage of DNA. The proposed mechanism of hydrolytic DNA cleavage has been discussed. MTT assay showed remarkable cytotoxicity on cervical cancer HeLa cell line and the IC50 values were 1.27, 4.13, and 3.92 µM for 1, 2 and 3, respectively, as compared to the IC50 value (13 µM) reported for cisplatin in HeLa cells. AO/PI and Annexin-V/PI assay suggest the induction of cell death primarily via apoptotic pathway. Nuclear staining using DAPI was used to assess changes in nuclear morphology during apoptotic cell death. The role of reactive oxygen species (ROS) for induction of apoptotic cell death was studied using H2DCF-DA assay and the result suggests that the generation of ROS by the complexes may be a possible cause for their antiproliferative activity. TUNEL assay showed DNA fragmentation in apoptotic cells. Cell cycle analysis using flow cytometry showed significant increase in the G2/M phase in HeLa cells by the compounds 1-3. Mononuclear Cu(II) complexes display remarkable cytotoxicity against cervical cancer HeLa cell line. The generation of ROS by the complexes may be a cause of their antiproliferative activity. Fluorescent images from DAPI staining assay revealed that the cells undergoing apoptosis displayed typical features like cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. TUNEL assay showed DNA fragmentation in apoptotic cells.

Modulation of risk of squamous cell carcinoma head and neck in North Indian population with polymorphisms in xeroderma pigmentosum complementation Group C gene.

BACKGROUND: Genetic variations in nucleotide excision repair genes can alter the risk of squamous cell carcinoma of head and neck (SCCHN). MATERIALS AND METHODS: The present study has genotyped 334 subjects from North Indian population for xeroderma pigmentosum complementation Group C (XPC) rs2228001A>C, XPC rs77907221 polyadenylate (PAT) deletion/insertion (D/I), xeroderma pigmentosum complementation Group D - rs13181A>C, and xeroderma pigmentosum complementation Type G rs17655 G>C polymorphisms with polymerase chain reaction (PCR)-restriction-fragment length polymorphism or allele-specific PCR methods. RESULTS: Compared to D allele, I allele for XPC PAT D/I polymorphism was associated with significantly decreased the risk of SCCHN (odds ratios = 0.67, 95% confidence interval [CI] =0.48-0.94, P = 0.03). Haplotype CI constituted from XPC polymorphisms was also associated with decreased risk of SCCHN (P = 0.004). In contrast, haplotype Crohn's disease significantly increased the risk for SCCHN (P < 0.00). A significant early onset of SCCHN was observed in individuals with CC genotype for XPC A>C polymorphism (P = 0.004). CONCLUSION: Our results suggest a possible risk modulation for SCCHN with XPC polymorphisms in North Indian population.

Anti-Leukemic Activity of Shikonin: Role of ERP57 in Shikonin Induced Apoptosis in Acute Myeloid Leukemia.

BACKGROUND/AIMS: ER-Stress and activation of unfolded protein response belong to the major factors involved in chemoresistance in cancer cells. In this study we investigated the effect of shikonin on the survival of acute myeloid leukemia cells and the role of ER-stress protein ERP57, a protein disulfide isomerase, in improvement of chemotherapy. METHODS: Using MTT assay we studied cytotoxic effects of shikonin on HL-60 cells. The flow cytometry was adopted to examine the shikonin induced mode of cell death in HL-60 cells. The overall protein expression alteration resulting from shikonin treatment was investigated using proteomics methods. Western blotting was performed to quantify the alteration in protein expression in HL-60 after shikonin treatment. Silencing and overexpression studies were carried out to highlight the therapeutic role of ERP57 in shikonin effect on AML cells. RESULTS: Shikonin induces apoptosis in HL-60 cells without significant effect on Primary cells from healthy volunteers. The apoptotic effect was dose and time dependent and was accompanied by strong alteration in cell proteome. Among the proteins targeted by shikonin, ERP57 was significantly downregulated in HL-60 after treatment. Compared to healthy control ERP57 was found to be highly expressed in AML cell line HL60 and was downregulated after shikonin treatment. Overexpression of ERP57 protected HL-60 from shikonin induced apoptosis, whereas knockdown of ERP57 expression resulted in increase in shikonin induced apoptosis. CONCLUSIONS: Our results demonstrate that ERP57 plays a crucial role in resistance towards shikonin induced apoptosis in AML cells. Targeting of ERP57 might offer a new therapeutic option for the treatment of acute myeloid leukemia.

The antioxidant protein PARK7 plays an important role in cell resistance to Cisplatin-induced apoptosis in case of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most malignant tumor in the adult kidney. Many factors are responsible for the development and progression of this tumor. Increased reactive oxygen species accumulation and altered redox status have been observed in cancer cells and this biochemical property of cancer cells can be exploited for therapeutic benefits. In earlier work we identified and characterize Protein DJ-1 (PARK7) as an oxidative stress squevenger in renal cells exposed to oxidative stress. To investigate whether the PARK7 or other oxidative stress proteins play a role in the renal cell carcinoma and its sensitivity or resistance to cytostatic drug treatment, differential proteomics analysis was performed with a cell model for clear cell renal carcinoma (Caki-2 and A498). Caki-2 cells were treated with cisplatin and differentially expressed proteins were investigated. The cisplatin treatment resulted in an increase in reactive oxygen species accumulation and ultimately apoptosis of Caki-2 and A498 cells. In parallel, the apoptotic effect was accompanied by a significant downregulation of antioxidant proteins especially PARK7. Knockdown of PARK7 using siRNA and overexpression using plasmid highlights the role of PARK7 as a key player in renal cell carcinoma response to cisplatin induced apoptosis. Overexpression of PARK7 resulted in significant decrease in apoptosis, whereas knockdown of the protein was accompanied by an increase in apoptosis in Caki-2 and A498 cells treated with cisplatin. These results highlights for the first time the important role of PARK7 in cisplatin induced apoptosis in clear renal cell carcinoma cells.

Controlled release of drug and better bioavailability using poly(lactic acid-co-glycolic acid) nanoparticles.

Tamoxifen (Tmx) embedded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-Tmx) is prepared to evaluate its better DNA cleavage potential, cytotoxicity using Dalton's lymphoma ascite (DLA) cells and MDA-MB231 breast cancer cells. PLGA-Tmx nanoparticles are prepared through emulsified nanoprecipitation technique with varying dimension of 17-30nm by changing the concentrations of polymer, emulsifier and drug. Nanoparticles dimension are measured through electron and atomic force microscopy. Interactions between tamoxifen and PLGA are verified through spectroscopic and calorimetric methods. PLGA-Tmx shows excellent DNA cleavage potential as compared to pure Tmx raising better bioavailability. In vitro cytotoxicity studies indicate that PLGA-Tmx reduces DLA cells viability up to ∼38% against ∼15% in pure Tmx. Hoechst stain is used to detect apoptotic DLA cells through fluorescence imaging of nuclear fragmentation and condensation exhibiting significant increase of apoptosis (70%) in PLGA-Tmx vis-à-vis pure drug (58%). Enhanced DNA cleavage potential, nuclear fragmentation and condensation in apoptotic cells confirm greater bioavailability of PLGA-Tmx as compared to pure Tmx in terms of receptor mediated endocytosis. Hence, the sustained release kinetics of PLGA-Tmx nanoparticles shows much better anticancer efficacy through enhanced DNA cleavage potential and nuclear fragmentation and, thereby, reveal a novel vehicle for the treatment of cancer.

Anti-cancer evaluation of quercetin embedded PLA nanoparticles synthesized by emulsified nanoprecipitation.

This study was carried out to synthesize quercetin (Qt) embedded poly(lactic acid) (PLA) nanoparticles (PLA-Qt) and to evaluate anti-cancer efficacy of PLA-Qt by using human breast cancer cells. PLA-Qt were synthesized by using novel emulsified nanoprecipitation technique with varying dimension of 32 ± 8 to 152 ± 9 nm of PLA-Qt with 62 ± 3% (w/w) entrapment efficiency by varying the concentration of polymer, emulsifier, drug and preparation temperature. The dimension of PLA-Qt was measured through transmission electron microscopy indicating larger particle size at higher concentration of PLA. The release rate of Qt from PLA-Qt was found to be more sustained for larger particle dimension (152 ± 9 nm) as compared to smaller particle dimension (32 ± 8 nm). Interaction between Qt and PLA was verified through spectroscopic and calorimetric methods. Delayed diffusion and stronger interaction in PLA-Qt caused the sustained delivery of Qt from the polymer matrix. In vitro cytotoxicity study indicate the killing of ∼ 50% breast cancer cells in two days at 100 µg/ml of drug concentration while the ∼ 40% destruction of cells require 5 days for PLA-Qt (46 ± 6 nm; 20mg/ml of PLA). Thus our results propose anticancer efficacy of PLA-Qt nanoparticles in terms of its sustained release kinetics revealing novel vehicle for the treatment of cancer.