Shiv Prakash Verma

Layered double hydroxides as effective carrier for anticancer drugs and tailoring of release rate through interlayer anions

Hydrophobic anticancer drug, raloxifene hydrochloride (RH) is intercalated into a series of magnesium aluminum layered double hydroxides (LDHs) with various charge density anions through ion exchange technique for controlled drug delivery. The particle nature of the LDH in presence of drug is determined through electron microscopy and surface morphology. The release of drug from the RH intercalated LDHs was made very fast or sustained by altering the exchangeable anions followed by the modified Freundlich and parabolic diffusion models. The drug release rate is explained from the interactions between the drug and LDHs along with order-disorder structure of drug intercalated LDHs. Nitrate bound LDH exhibits greater interaction with drug and sustained drug delivery against the loosely interacted phosphate bound LDH-drug, which shows fast release. Cell viability through MTT assay suggests drug intercalated LDHs as better drug delivery vehicle for cancer cell line against poor bioavailability of the pure drug. In vivo study with mice indicates the differential tumor healing which becomes fast for greater drug release system but the body weight index clearly hints at damaged organ in the case of fast release system. Histopathological experiment confirms the damaged liver of the mice treated either with pure drug or phosphate bound LDH-drug, fast release system, vis-à-vis normal liver cell morphology for sluggish drug release system with steady healing rate of tumor. These observations clearly demonstrate that nitrate bound LDH nanoparticle is a potential drug delivery vehicle for anticancer drugs without any side effect.

In situ grafted nanostructured ZnO/carboxymethyl cellulose nanocomposites for efficient delivery of curcumin to cancer

In this present manuscript, zinc oxide (ZnO) nanoparticles embedded carboxymethyl cellulose (CMC) bionanocomposite were prepared by in situ grafting and the hydrophobic anticancer drug curcumin (Cur) was loaded into it. Structural, morphological, and physiochemical behavior of prepared curcumin-loaded CMC/ZnO nanocomposites (NCs) were characterized by FTIR, XRD, SEM, TEM, TGA, and DTA. The drug entrapment efficiency was evaluated and the in vitro efficacy as anticancer drug delivery vehicle was analyzed. The potential toxicity of curcumin-loaded ZnO/CMC NCs (Cur/ZnO/CMC NCs) was studied by using L929 and MA104 cell lines via MTT assay. The cellular uptake study of Cur/ZnO/CMC NCs by normal (L929) and cancer (MA104) cells carried out by using ethanol extraction and by FACS analysis has been reported. The results of this investigation demonstrate that the nanomatrix synthesized can effectively deliver the anticancer drug curcumin, and hence appears to be a promising nanoformulation for anticancer therapy and other biomedical applications.

Asparagus racemosus leaf extract inhibits growth of UOK 146 renal cell carcinoma cell line: simultaneous oncogenic PRCCTFE3 fusion transcript inhibition and apoptosis independent cell death.

AIMS To evaluate anti-cancer activity of Asparagus racemosus (AR) leaf extract on UOK146, a renal cell carcinoma cell line, and explore its mechanism of action. MATERIALS AND METHODS Dried AR leaves were extracted with chloroform and dissolved in DMSO. This extract was applied to UOK146 and cell death was estimated by MTT assay. In addition PRCC-TFE3 fusion transcripts were detected by real time PCR. RESULTS Extract was found to be cytotoxic with an IC50 of 0.9 mg/ml as estimated by dose response curve. Antitumor activity of the permissible doses of the extract was assessed by the down regulation of PRCC-TFE3 fusion transcript (38%) responsible for oncogenicity of the UOK146 cell line. No increment in the BAX, a proapoptotic marker level was observed. CONCLUSIONS Evidence of antiproliferative effect, PRCC-TFE3 fusion transcript inhibition and static BAX level clearly indicate that AR extract provides or elicits an apoptosis independent anticancer effect on RCC cells by some specific mechanism of regulation.

Aqueous Extract of Anticancer Drug CRUEL Herbomineral Formulation Capsules Exerts Anti-proliferative Effects in Renal Cell Carcinoma Cell Lines.

PURPOSE Anti-cancer activity evaluation of aqueous extract of CRUEL (herbomineral formulation) capsules on renal cell carcinoma cell lines, and exploration of mechanisms of cell death. MATERIALS AND METHODS To detect the cytotoxic dose concentration in renal cell carcinoma (RCC) cells, MTT assays were performed and morphological changes after treatment were observed by inverted microscopy. Drug effects against RCC cell lines were assessed with reference to cell cycle distribution (flow cytometry), anti-metastatic potential (wound healing assay) and autophagy(RT-PCR). RESULTS CRUEL showed anti-proliferative effects against RCC tumor cell lines with an IC50 value of approximately 4mg/mL in vitro, while inducing cell cycle arrest at S-phase of the cell cycle and inhibiting wound healing. LC3 was found to be up-regulated after drug treatment by RT-PCR resulting in an autophagy mode of cell death. CONCLUSIONS This study provides experimental validation for antitumor activity of CRUEL.

Novel splicing in IGFN1 intron 15 and role of stable G-quadruplex in the regulation of splicing in renal cell carcinoma

The IGFN1 (Immunoglobulin-Like And Fibronectin Type III Domain Containing 1) gene has a role in skeletal muscle function and is also involved in metastatic breast cancer, and the isoforms with three N-terminal globular domains are sufficient for its function in skeletal muscle. Two novel splicing isoforms of IGFN1 have been identified in renal cell carcinoma (RCC), one with 5’exon extension and an isoform with a novel exon. The role of G-quadruplex, a non-B DNA, was explored for the splicing alteration of IGFN1 in RCC. G-quadruplexes are the secondary structures acquired by stacking of G-quartets by Hoogsteen hydrogen bonding in DNA and RNA. IGFN1 has intronic potential G-quadruplex forming sequence (PQS) folding into G-quadruplex and is studied for its involvement in aberrant splicing. A PQS in the intron 15 of IGFN1 gene was observed in our in silico analysis by QGRS mapper and non BdB web servers. We observed PQS folds into stable G-quadruplex structure in gel shift assay and circular dichroism (CD) spectroscopy in the presence of G-quadruplex stabilizing agents Pyridostatin (PDS) and KCl, respectively. G-quadruplex formation site with single base resolution was mapped by Sanger sequencing of the plasmid constructs harbouring the cloned PQS and its mutant. This stable G-quadruplex inhibits reverse transcriptase and taq polymerase in reverse transcriptase & PCR stop assays. PDS changes the different splicing isoforms of IGFN1 in UOK146 cell line, displaying involvement of intronic G-quadruplex in IGFN1 splicing. These results lead us to propose that a stable G-quadruplex structure is formed in IGFN1 intron and a reason behind IGFN1 aberrant splicing which could be targeted for therapeutic intervention.

Sodium butyrate induces cell death by autophagy and reactivates a tumor suppressor gene DIRAS1 in renal cell carcinoma cell line UOK146

Sodium butyrate (SB), a histone deacetylase inhibitor, is emerging as a potent anti-cancer drug for different types of cancers. In the present study, anti-cancer activity of SB in Xp11.2 (TFE3) translocated renal cell carcinoma cell line UOK146 was studied. Anti-proliferative effect of SB in renal cell carcinoma (RCC) cell line UOK146 was evaluated by MTT assay and morphological characteristics were observed by phase contrast microscopy which displayed the cell death after SB treatment. SB induces DNA fragmentation and change in nuclear morphology observed by increased sub-G1 region cell population and nuclear blebbings. Cell cycle arrest at G2/M phase was found after SB treatment. UOK146 cell line shows autophagy mode of cell death as displayed by acridine orange staining and flow cytometry analysis. LC3-II, a protein marker of autophagy, was also found to be upregulated after SB treatment. A tumor suppressor gene DIRAS1 was upregulated after SB treatment, displaying its anti-cancer potential at molecular level. These findings suggest that SB could serve as a novel regulator of tumor suppressors and lead to the discovery of novel therapeutics with better and enhanced anti-cancer activity.