Girish Mahajan

Development and evaluation of folate functionalized albumin nanoparticles for targeted delivery of gemcitabine

Gemcitabine is one of the most potent anticancer agents acting on a wide range of solid tumors, however, its use is limited by short half life and high dose leading to serious side effects. The present investigation describes the development and characterization of folate functionalized gemcitabine loaded bovine serum albumin nanoparticles (Fa-Gem-BSANPs). The nanoparticles were prepared by desolvation cross-linking technique and characterized for various parameters including morphology, particle size, zeta potential, drug loading and release profile. The particle size of Gem-BSANPs and Fa-Gem-BSANPs was found to be 159.1±5.29 and 208.7±1.80 nm, respectively. DSC and XRD analysis indicated amorphous nature of the drug within the particles. The encapsulated gemcitabine exhibited less hemolytic properties as compared to native drug. The anticancer activity of Fa-Gem-BSANPs was evaluated in folate receptor over expressing cell lines (Ovcar-5 and MCF-7) and folate receptor deficient cell line (MIAPaCa-2). The Fa-Gem-BSANPs showed superior anticancer activity as compared to Gem-BSANPs in Ovcar-5 and MCF-7 cells while no significant difference in cytotoxicity was found with MIAPaCa-2 cells. Confocal microscopy indicated facilitated intracellular uptake of Fa-Gem-BSANPs in MCF-7, which in turn result in a higher potential for apoptosis. Further, Fa-Gem-BSANPs exhibited improved anti-tumor activity in Ehrlich solid tumor model in mice. In conclusion, our study indicates that folate functionalized nanoparticles confer enhance cellular uptake and cytotoxicity for gemcitabine.

Quinomycin A targets Notch signaling pathway in pancreatic cancer stem cells

Cancer stem cells (CSCs) appear to explain many aspects of the neoplastic evolution of tumors and likely account for enhanced therapeutic resistance following treatment. Dysregulated Notch signaling, which affects CSCs plays an important role in pancreatic cancer progression. We have determined the ability of Quinomycin to inhibit CSCs and the Notch signaling pathway. Quinomycin treatment resulted in significant inhibition of proliferation and colony formation in pancreatic cancer cell lines, but not in normal pancreatic epithelial cells. Moreover, Quinomycin affected pancreatosphere formation. The compound also decreased the expression of CSC marker proteins DCLK1, CD44, CD24 and EPCAM. In addition, flow cytometry studies demonstrated that Quinomycin reduced the number of DCLK1+ cells. Furthermore, levels of Notch 1–4 receptors, their ligands Jagged1, Jagged2, DLL1, DLL3, DLL4 and the downstream target protein Hes-1 were reduced. The γ-secretase complex proteins, Presenilin 1, Nicastrin, Pen2, and APH-1, required for Notch activation also exhibited decreased expression. Ectopic expression of the Notch Intracellular Domain (NICD) partially rescued the cells from Quinomycin mediated growth suppression. To determine the effect of Quinomycin on tumor growth in vivo, nude mice carrying tumor xenografts were administered Quinomycin intraperitoneally every day for 21 days. Treatment with the compound significantly inhibited tumor xenograft growth, coupled with significant reduction in the expression of CSC markers and Notch signaling proteins. Together, these data suggest that Quinomycin is a potent inhibitor of pancreatic cancer that targets the stem cells by inhibiting Notch signaling proteins.

Anticancer activity of gallic acid template-based benzylidene indanone derivative as microtubule destabilizer.

Benzylidene indanones have been designed and synthesized from gallic acid, a plant phenolic acid as possible anticancer agent. The best analogue of the series, that is, 3‐(3′,4′,5′‐trimethoxyphenyl)‐4,5,6‐trimethoxy‐2‐(4˝‐nitrobenzylidene)‐indan‐1‐one (8) exhibited potent cytotoxicity (IC50=3–10 μm) against several human cancer cell lines through microtubule destabilization (IC50=1.54 μm) after occupying colchicine‐binding site of β‐tubulin. In cell cycle analysis, compound 8 exerted G2/M phase arrest in both MCF‐7 and MDA‐MB‐231 cells and induced apoptosis. It reduced 34.8% solid tumor in in vivo Ehrlich ascite carcinoma in Swiss albino mice at 30 mg/kg dose. In acute oral toxicity experiment, it was tolerable up to 300 mg/kg doses in Swiss albino mice. The lead compound 8 needs to be optimized for better activity.

Fermentation, Isolation, Structure, and antidiabetic activity of NFAT-133 produced by Streptomyces strain PM0324667

Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound.

In Vitro and In Vivo Activities of Antibiotic PM181104

ABSTRACT Drug resistance has become a global threat that, if not addressed, may return us to the preantibiotic era. A way to overcome the problem of growing incidence of global antibiotic resistance is to introduce compounds belonging to classes that are new to the clinic. During a screening of the marine microbial extract library for new antibiotics, one of the extracts showed promising antibacterial activity against Gram-positive organisms. Bioactivity-guided isolation and characterization of active metabolites led to the discovery of a novel thiazolyl cyclic-peptide antibiotic, PM181104. It was isolated and characterized from a marine sponge-associated actinobacterium strain of the genus Kocuria (MTCC 5269). The compound exhibited a potent in vitro antibacterial activity against a broad range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The MIC values evaluated for the compound were found to be in the single-digit nanomolar range. In in vivo studies of PM181104 in a BALB/c murine septicemia model, the compound displayed 100% effective dose (ED100) values of 2.5 and 5.0 mg/kg of body weight against MRSA and 10.0 mg/kg against VRE. In this report, in vitro and in vivo studies of PM181104 are described.

In Vitro and In Vivo Anticancer Activity of Root Extracts of Sansevieria liberica Gerome and Labroy (Agavaceae)

Introduction. Sansevieria liberica Gerome and Labroy (Agavaceae) is a perennial plant widely distributed in tropical Africa. Preparations of the plant are commonly used across Nigeria for the treatment of inflammatory conditions. Based on the fact that herbal medicine is a strong component of integrative medicine, this study was conducted to evaluate the anticancer activity of root extracts of Sansevieria liberica. Methods. Sulforhodamine B (SRB) in vitro cytotoxicity assay, Sarcoma-180 (S-180) ascites and solid tumor, and L1210 lymphoid leukemia in vivo models were used in this study. Results. SL-A002 (IC50 23 µg/mL with HeLa), SL-A003 (IC50 22 µg/mL with HCT-116), and SL-A004 (IC50 23 and 18 µg/mL with A549 and THP-1, resp.) demonstrated significant activity in the SRB cytotoxicity assay. Potency was highest with the following pairs of extract : cancer cell line: SL-A002 : HeLa (IC50 23 µg/mL), SL-A003 : HCT-116 (IC50 22 µg/mL), and SL-A004 : THP-1 (IC50 18 µg/mL). SL-A002 demonstrated significant dose-dependent antitumor activity in the Sarcoma-180 (S-180) ascites model with peak effect produced at the dose of 120 mg/kg (i.p.) with inhibition of 89.36% compared to 97.96% for 5-FU (20 mg/kg i.p.). The inhibition of tumor growth by SL-A002 in the S-180 solid tumor model was 47.40% compared to a value of 50.18% for 5-FU. SL-A002 was also significantly active in the L1210 lymphoid leukemia model with 158.33% increase in mean survival time, the same value for 5-FU. Conclusions. The hydroethanolic extract of Sansevieria liberica, SL-A002, possesses significant anticancer activity to warrant further extensive study to identify, isolate, and characterize the specific bioactive molecules responsible for the observed antitumor activity and the precise mechanism(s) of action.

Long-circulatory nanoparticles for gemcitabine delivery: Development and investigation of pharmacokinetics and in-vivo anticancer efficacy

The anticancer potential of gemcitabine, a nucleoside analog, is compromised due to the enzymatic degradation into inactive form leading to the short half-life in systemic circulation. Novel delivery strategies are required to improve therapeutic efficacy of this potential drug. Monomethoxy polyethylene glycol amine-polylactide-co-glycolide (mPEG-PLGA) co-polymer was synthesized and characterized by FTIR and (1)H NMR. Gemcitabine loaded mPEG-PLGA nanoparticles (NPs) were developed and investigated for pharmacokinetic profile and in vivo anticancer activity. The mPEG-PLGA NPs (size: 267±10nm, zeta potential: -17.5±0.2mV) exhibited sustained drug release profile and were found to be compatible with blood. The mPEG-PLGA NPs were able to evade the uptake by macrophages (i.e. THP-1 and J774A) by reducing the adsorption of proteins on the surface of NPs. The enhanced cellular uptake and cell cytotoxicity was observed by mPEG-PLGA NPs in MiaPaCa-2 and MCF-7 cells. The half-life of gemcitabine in mPEG-PLGA NPs was remarkably enhanced (19 folds) than native gemcitabine. Further, the pharmacokinetic modulation of gemcitabine using mPEG-PLGA-NPs was translated in improved anticancer efficacy as compared to native gemcitabine in Ehrlich ascites bearing Balb-c mice. The results demonstrated the potential of long-circulatory nanoparticles in improving the pharmacokinetic profile and in-turn the anticancer efficacy of gemcitabine.

A novel colchicine-based microtubule inhibitor exhibits potent antitumor activity by inducing mitochondrial mediated apoptosis in MIA PaCa-2 pancreatic cancer cells.

Colchicine, an antimitotic alkaloid isolated from Colchicum autumnale, is a classical drug for treatment of gout and familial Mediterranean fever. It causes antiproliferative effects through the inhibition of microtubule formation, which leads to mitotic arrest and cell death by apoptosis. Here, we report that a novel colchicine analog, 4o (N-[(7S)-1,2,3-trimethoxy-9-oxo-10-[3-(trifluoromethyl)-4-chlorophenylamino]-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide), which exhibited potent anticancer activities both in vitro and in vivo. In this study, 4o with excellent pharmacokinetic profile and no P-gp induction liability displayed strong inhibition of proliferation against various human cancer cell lines. However, pancreatic cancer cell line MIA PaCa-2 was found to be more sensitive towards 4o and showed strong inhibition in concentration and time-dependent manner. By increasing intracellular reactive oxygen species (ROS) levels, 4o induced endoplasmic reticular stress and mitochondrial dysfunction in MIA PaCa-2 cells. Blockage of ROS production reversed 4o-induced endoplasmic reticulum (ER) stress, calcium release, and cell death. More importantly, it revealed that increased ROS generation might be an effective strategy in treating human pancreatic cancer. Further 4o treatment induced mitotic arrest, altered the expression of cell cycle-associated proteins, and disrupted the microtubules in MIA PaCa-2 cells. 4o treatment caused loss of mitochondrial membrane potential, cytochrome c release, upregulation of Bax, downregulation of Bcl-2, and cleavage of caspase-3, thereby showing activation of mitochondrial mediated apoptosis. The in vivo anticancer activity of the compound was studied using sarcoma-180 (ascitic) and leukemia (P388 lymphocytic and L1210 lymphoid) models in mice and showed promising antitumor activity with the least toxicity unlike colchicine. Such studies have hitherto not been reported. Taken together, these findings highlighted that 4o, a potent derivative of colchicine, causes tumor regression with reduced toxicity and provides a novel anticancer candidate for the therapeutic use.

PLGA nanoparticles augmented the anticancer potential of pentacyclic triterpenediol in vivo in mice

A pentacyclic triterpenediol (TPD) from Boswellia serrata exhibited a good anticancer potential preclinically, however, it has low aqueous solubility and high lipophilicity, which therefore, necessitate suitable formulation development for in vivo application. In the present study TPD-loaded PLGA nanoparticles (TPD NPs) were prepared by an emulsion–diffusion–evaporation technique which exhibited an average particle size in the order of about 161 nm as confirmed by dynamic light scattering (DLS) and atomic force microscopy (AFM). The thermal analysis confirms that the TPD was entrapped into the NPs in an amorphous form. In vitro cell culture experiments indicated higher cellular cytotoxicity of the TPD-loaded NPs over free TPD in MCF-7 and OVCAR-5 cells. The higher cytotoxicity of TPD NPs was attributed to enhanced cellular apoptosis, loss of membrane potential and generation of high reactive oxygen species (ROS). The TPD-loaded NPs demonstrated a significantly higher in vivo anticancer potential as compared to TPD solution in the Ehrlich ascites tumor (EAT) model following intraperitoneal administration. Furthermore, no hematological and biochemical toxicity in EAT bearing mice was observed after the treatment. The results showed that the developed PLGA-NPs could be a potential option for improved TPD delivery in cancer chemotherapy.

Anticancer activity of Aristolochia ringens Vahl. (Aristolochiaceae).

Cancer is a leading cause of death worldwide and sustained focus is on the discovery and development of newer and better tolerated anticancer drugs especially from plants. The sulforhodamine B (SRB) in vitro cytotoxicity assay, sarcoma-180 (S-180) ascites and solid tumor, and L1210 lymphoid leukemia in vivo models were used to investigate the anticancer activity of root extracts of Aristolochia ringens Vahl. (Aristolochiaceae; 馬兜鈴 mǎ dōu líng). AR-A001 (IC50 values of 20 μg/mL, 22 μg/mL, 3 μg/mL, and 24 μg/mL for A549, HCT-116, PC3, and THP-1 cell lines, respectively), and AR-A004 (IC50 values of 26 μg/mL, 19.5 μg/mL, 12 μg/mL, 28 μg/mL, 30 μg/mL, and 22 μg/mL for A549, HCT-116, PC3, A431, HeLa, and THP-1, respectively), were observed to be significantly active in vitro. Potency was highest with AR-A001 and AR-A004 for PC3 with IC50 values of 3 μg/mL and 12 μg/mL, respectively. AR-A001 and AR-A004 produced significant (p < 0.05–0.001) dose-dependent inhibition of tumor growth in the S-180 ascites model with peak effects produced at the highest dose of 120 mg/kg. Inhibition values were 79.51% and 89.98% for AR-A001 and AR-A004, respectively. In the S-180 solid tumor model, the inhibition of tumor growth was 29.45% and 50.50% for AR-A001 (120 mg/kg) and AR-A004 (110 mg/kg), respectively, compared to 50.18% for 5-fluorouracil (5-FU; 20 mg/kg). AR-A001 and AR-A004 were also significantly active in the leukemia model with 211.11% and 155.56% increase in mean survival time (MST) compared to a value of 211.11% for 5-FU. In conclusion, the ethanolic (AR-A001) and dichloromethane:methanol (AR-A004) root extracts of AR possess significant anticancer activities in vitro and in vivo.