Deepak Chitkara

Chemoresistance in Prostate Cancer Cells Is Regulated by miRNAs and Hedgehog Pathway

Many prostate cancers relapse due to the generation of chemoresistance rendering first-line treatment drugs like paclitaxel (PTX) ineffective. The present study aims to determine the role of miRNAs and Hedgehog (Hh) pathway in chemoresistant prostate cancer and to evaluate the combination therapy using Hh inhibitor cyclopamine (CYA). Studies were conducted on PTX resistant DU145-TXR and PC3-TXR cell lines and clinical prostate tissues. Drug sensitivity and apoptosis assays showed significantly improved cytotoxicity with combination of PTX and CYA. To distinguish the presence of cancer stem cell like side populations (SP), Hoechst 33342 flow cytometry method was used. PTX resistant DU145 and PC3 cells, as well as human prostate cancer tissue possess a distinct SP fraction. Nearly 75% of the SP cells are in the G0/G1 phase compared to 62% for non-SP cells and have higher expression of stem cell markers as well. SP cell fraction was increased following PTX monotherapy and treatment with CYA or CYA plus PTX effectively reduced their numbers suggesting the effectiveness of combination therapy. SP fraction cells were allowed to differentiate and reanalyzed by Hoechst staining and gene expression analysis. Post differentiation, SP cells constitute 15.8% of total viable cells which decreases to 0.6% on treatment with CYA. The expression levels of P-gp efflux protein were also significantly decreased on treatment with PTX and CYA combination. MicroRNA profiling of DU145-TXR and PC3-TXR cells and prostate cancer tissue from the patients showed decreased expression of tumor suppressor miRNAs such as miR34a and miR200c. Treatment with PTX and CYA combination restored the expression of miR200c and 34a, confirming their role in modulating chemoresistance. We have shown that supplementing mitotic stabilizer drugs such as PTX with Hh-inhibitor CYA can reverse PTX chemoresistance and eliminate SP fraction in androgen independent, metastatic prostate cancer cell lines.

miRNA profiling in pancreatic cancer and restoration of chemosensitivity

Pancreatic cancers relapse due to small but distinct population of cancer stem cells (CSCs) which are in turn regulated by miRNAs. The present study identifies a series of miRNAs which were either upregulated (e.g. miR-146) or downregulated (e.g. miRNA-205, miRNA-7) in gemcitabine resistant MIA PaCa-2 cancer cells and clinical metastatic pancreatic cancer tissues. Gemcitabine resistant MIA PaCa-2 cells possessed distinct ALDH-positive CSC fraction expressing stem cell markers OCT3/4 and CD44 and chemoresistance marker class IIIβ-tubulin (TUBB3) which decreases on transfection with miR-205 resulting in the restoration of chemosensitivity to gemcitabine.

Efficacy of gemcitabine conjugated and miRNA-205 complexed micelles for treatment of advanced pancreatic cancer.

Clinical effectiveness of gemcitabine in pancreatic cancer is hindered due to its rapid plasma metabolism and development of chemo-resistance. We have previously delineated the significant role of miRNAs in mediating the growth and proliferation of cancer stem cells (CSCs) which in turn result in chemo-resistance, invasion and metastasis. Here, we designed self-assembling, gemcitabine conjugated cationic copolymers for co-delivery of a tumor suppressor miRNA-205 (miR-205) and evaluated their in vivo efficacy in a pancreatic cancer ectopic tumor model developed using gemcitabine resistant MIA PaCa-2(R) cells. Combination formulations showed mean a particle size of <100 nm and gemcitabine payload of >10% w/w, exhibited miRNA complexation at N/P ratio of 4/1, sustained release of gemcitabine for >10 days, transfection efficiency of >90%, extended miRNA and drug stability in serum. Functional assays in gemcitabine resistant MIA PaCa-2(R) and CAPAN-1(R) pancreatic cancer cells revealed that the combination formulations effectively reversed chemo-resistance, invasion and migration. In pancreatic tumor model, the combination formulation treated group showed significant inhibition of tumor growth. Immuno-hiostochemical analysis revealed decreased tumor cell proliferation with increased apoptosis in the animals treated with miR-205 and gemcitabine combination.

Self-assembling, amphiphilic polymer-gemcitabine conjugate shows enhanced antitumor efficacy against human pancreatic adenocarcinoma.

The therapeutic efficacy of gemcitabine is severely compromised due to its rapid plasma metabolism. Moreover, its hydrophilicity poses a challenge for its efficient entrapment in nanosized delivery systems and to provide a sustained release profile. In this study, gemcitabine was covalently conjugated to poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (PEG-PCC) which could self-assemble into micelles of 23.6 nm. These micelles afforded protection to gemcitabine from plasma metabolism as evident by negligible amount of gemcitabine and its metabolite dFdU detected in the plasma after 24 h. A controlled release of gemcitabine from the micelles was observed with 53.89% drug release in 10 days in the presence of protease enzyme Cathepsin B. Gemcitabine conjugated micelles were cytotoxic, showed internalization, and induced cell apoptosis in MIA PaCa-2 and L3.6pl pancreatic cancer cell lines. These micelles efficiently inhibited tumor growth when injected intravenously into MIA PaCa-2 cell derived xenograft tumor bearing NSG mice at a dose of 40 mg/kg in terms of reduced tumor volume and tumor weight (0.38 g vs 0.58 g). TUNEL assay revealed that gemcitabine conjugated micelles induced a much higher extent of apoptosis in the tumor tissues compared to free gemcitabine. In conclusion, gemcitabine conjugated micelles were able to enhance the drug payload, protect it from rapid plasma metabolism, and provide a sustained release and showed enhanced antitumor activity, and thus have the potential to provide a better therapeutic alternative for treating pancreatic cancer.

Polyanhydrides as localized drug delivery carrier: an update

Background: There is a continuing thrust to increase the efficacy and reduce the toxicity of existing and new drug molecules for their better usage to treat disease. Localized drug delivery has been explored in the same way, which can provide a platform to target local diseased tissues and can reduce the burden on the body by reducing the dose size and hence the dose-related toxicity of the molecules. Various polymers have evolved for the purpose of localized drug delivery, however, polyanhydrides are considered the best, supported by products in the clinical phases. Objective: To demonstrate the advantages of localized delivery using basic concepts and describing polyanhydride carrier with products such as Gliadel® and Septacin™. Methods: The rationale behind localized drug delivery and the carrier for the same are dealt with. Polyanhydrides discussed in detail are those from subclasses that have been given less emphasis previously and have been developed or investigated in the last 5 years. Results/conclusion: From the recent update on polyanhydrides, it can be concluded that these polymers have great potential as localized drug delivery carriers due to the versatility of their properties. However, the quest to stabilize the system in order to achieve a long shelf life remains ongoing.

miRNAs in pancreatic cancer: therapeutic potential, delivery challenges and strategies.

Pancreatic ductal adenocarcinoma (PDAC) is a severe pancreatic malignancy and is predicted to victimize 1.5% of men and women during their lifetime (Cancer statistics: SEER stat fact sheet, National Cancer Institute, 2014). miRNAs have emerged as a promising prognostic, diagnostic and therapeutic tool to fight against pancreatic cancer. miRNAs could modulate gene expression by imperfect base-pairing with target mRNA and hence provide means to fine-tune multiple genes simultaneously and alter various signaling pathways associated with the disease. This exceptional miRNA feature has provided a paradigm shift from the conventional one drug one target concept to one drug multiple target theory. However, in vivo miRNA delivery is not fully realized due to challenges posed by this special class of therapeutic molecules, which involves thorough understanding of the biogenesis and physicochemical properties of miRNA and delivery carriers along with the pathophysiology of the PDAC. This review highlights the delivery strategies of miRNA modulators (mimic/inhibitor) in cancer with special emphasis on PDAC since successful delivery of miRNA in vivo constitutes the major challenge in clinical translation of this promising class of therapeutics.

Micellar Delivery of Cyclopamine and Gefitinib for Treating Pancreatic Cancer

Hedgehog (Hh) and epidermal growth factor receptor (EGFR) signaling are involved in pancreatic cancer progression. Targeting these pathways simultaneously with cyclopamine (Hh inhibitor) and gefitinib (EGFR inhibitor) is a promising approach for treating pancreatic cancer. However, the major limitation for effective clinical translation of these molecules is their low aqueous solubility. We have previously demonstrated that methoxy polyethyleneglycol-b-poly(carbonate-co-lactic acid) {mPEG-b-P(CB-co-LA)} copolymer solubilizes hydrophobic anticancer drugs and has the potential to deliver to tumors by an enhanced permeability and retention (EPR) effect. In this study, using the nanoprecipitation method, cyclopamine and gefitinib were efficiently loaded into mPEG-b-P(CB-co-LA) micelles with encapsulation efficiencies of 94.4 and 88.6%, respectively. These micelles had a narrow particle size distribution with a mean particle size of 54.3 nm and a PDI of 0.14. Combination therapy showed a synergistic effect against L3.6pl cells but an additive effect against MIA PaCa-2 cells. Caspase 3/7 activity was also increased when this combination therapy was used, indicating apoptotic cell death. Gene and protein expression analysis indicated cross-talk between Hh and EGFR signaling. Furthermore, the combination decreased tumor growth rate in L3.6pl-derived xenograft mouse tumors. These data suggest the applicability of our micellar system to effectively load and deliver cyclopamine and gefitinib for combination chemotherapy.

Chitosan-modified PLGA polymeric nanocarriers with better delivery potential for tamoxifen

Breast cancer is believed as the second most common cause of cancer-related deaths in women for which tamoxifen is frequently prescribed. Despite many promises, tamoxifen is associated with various challenges like low hydrophilicity, poor bioavailability and dose-dependent toxicity. Therefore, it was envisioned to develop tamoxifen- loaded chitosan-PLGA micelles for potential safe and better delivery of this promising agent. The chitosan-PLGA copolymer was synthesised and characterised by Fourier Transform-Infrared, Ultraviolet-visible and Nuclear Magnetic Resonance spectroscopic techniques. The drug-loaded nanocarrier was characterised for drug-pay load, micrometrics, surface charge and morphological attributes. The developed system was evaluated for in-vitro drug release, haemolytic profile, cellular-uptake, anticancer activity by cytotoxicity assay and dermatokinetic studies. The developed nano-system was able to substantially load the drug and control the drug release. The in-vitro cytotoxicity offered by the system was significantly enhanced vis-a-vis plain drug, and there was no substantial haemolysis. The IC50 values were significantly decreased and the nanocarriers were uptaken by MCF-7 cells, noticeably. The carrier was able to locate the drug in the interiors of rat skin in considerable amounts to that of the conventional product. This approach is promising as it provides a biocompatible and effective option for better delivery of tamoxifen.

Nanocarrier-based co-delivery of small molecules and siRNA/miRNA for treatment of cancer

Aberrant gene expression can trigger several vital molecular events that not only result in carcinogenesis but also cause chemoresistance, metastasis and relapse. Gene-based therapies using siRNA/miRNA have been suggested as new treatment method to improve the current regimen. Although these agents can restore the normal molecular cascade thereby resensitizing the cancer cells, delivering a standard regimen (either subsequently or simultaneously) is necessary to achieve the therapeutic benefit. However, co-delivery using a single carrier could give an additional advantage of similar biodistribution profile of the loaded agents. While much research has been carried out in this field in recent years, challenges involved in designing combination formulations including efficient coloading, stability, appropriate biodistribution and target specificity have hampered their clinical translation. This article highlights current aspects of nano-carriers used for co-delivery of small molecules and genes to treat cancer.

Structural modifications in polymeric micelles to impart multifunctionality for improved drug delivery

Polymeric micelles are macromolecular nanoconstructs which are formed by self-assembly of synthetic amphiphilic block copolymers. These copolymers could be chemically modified to expand their functionality and hence obtain a multifunctional micelle which could serve several functions simultaneously, for example, long circulation time along with active targeting, smart polymeric micelles providing on-demand drug release for example, pH responsive micelles, redox- and light-sensitive micelles, charge-conversion micelles and core/shell cross-linked micelles. Additionally, micelles could be tailored to carry a contrast agent or siRNA/miRNA along with the drug for greater clinical benefit. The focus of the current commentary would be to highlight such chemical modifications which impart multifunctionality to a single carrier and discuss challenges involved in clinical translation of these multifunctional micelles.