Pharmacological attenuation of melanoma by tryptanthrin pertains to the suppression of MITF-M through MEK/ERK signaling axis.

Melanoma is the most aggressive among all types of skin cancers. The current strategies against melanoma utilize BRAFV600E, as a focal point for targeted therapy. However, therapy resistance developed in melanoma patients against the conventional anti-melanoma drugs hinders the ultimate benefits of targeted therapies. A major mechanism by which melanoma cells attain therapy resistance is via the activation of microphthalmia-associated transcription factor-M (MITF-M), the key transcription factor and oncogene aiding the survival of melanoma cells. We demonstrate that tryptanthrin (Tpn), an indole quinazoline alkaloid, which we isolated and characterized from Wrightia tinctoria, exhibits remarkable anti-tumor activity towards human melanoma through the down-regulation of MITF-M. Microarray analysis of Tpn-treated melanoma cells followed by a STRING protein association network analysis revealed that differential expression of genes in melanoma converges at MITF-M. Furthermore, in vitro and in vivo studies conducted using melanoma cells with differential MITF-M expression status, endogenously or ectopically, demonstrated that the anti-melanoma activity of Tpn is decisively contingent on its efficacy in down-regulating MITF-M expression. Tpn potentiates the degradation of MITF-M via the modulation of MEK1/2-ERK1/2-MITF-M signaling cascades. Murine models demonstrate the efficacy of Tpn in attenuating the migration and metastasis of melanoma cells, while remaining pharmacologically safe. In addition, Tpn suppresses the expression of mutated BRAFV600E and inhibits Casein Kinase 2α, a pro-survival enzyme that regulates ERK1/2 homeostasis in many tumor types, including melanoma. Together, we point to a promising anti-melanoma drug in Tpn, by virtue of its attributes to impede melanoma invasion and metastasis by attenuating MITF-M.

Pre-clinical evidences for the efficacy of tryptanthrin as a potent suppressor of skin cancer.

OBJECTIVE: Clinical trials have demonstrated the efficacy of indigo naturalis, a traditional Chinese medicine ingredient, against psoriasis, a skin disease characterized by keratinocyte hyperproliferation and inflammation. The present study investigates the efficacy of tryptanthrin, a bioactive compound in indigo naturalis, against non-melanoma skin cancer (NMSC) and the signalling events involved. METHODS: Efficacy of tryptanthrin against NMSC was assessed using DMBA/PMA-induced skin carcinogenesis model in Swiss albino mice. Immunostaining for PCNA and ki-67 was used to mark proliferating cells in tissues. Haematoxylin and eosin staining and toluidine staining were employed to assess inflammation, and TUNEL assay was used to detect apoptosis in tissues. The signalling events were evaluated using Western blot, imunohistochemistry and immunofluorescence staining. MTT assay and clonogenic assay were performed to assess the viability and proliferation of cancer cells, in vitro. RESULTS: In mice, topical application of tryptanthrin suppressed skin carcinogenesis. It attenuated inflammation, impeded the proliferation of hair follicle (HF) cells and suppressed the activation of ß-catenin, a major driver of HF cell proliferation. Additionally tryptanthrin suppressed the activation of ERK1/2 and p38, both of which promote ß-catenin activation and lowered the expression of c-Myc and cyclin-D1. Tryptanthrin suppressed the proliferation of the human NMSC cell line, A431 and abrogated EGF-induced activation of ß-catenin and subsequent cytoskeletal rearrangement. CONCLUSION: The study demonstrates with molecular evidence that tryptanthrin is an effective suppressor of NMSC.

Chitosan Encapsulation Enhances the Bioavailability and Tissue Retention of Curcumin and Improves its Efficacy in Preventing B[a]P-induced Lung Carcinogenesis.

The rate of lung cancer incidence is alarmingly mounting, despite the decline of smoking and tobacco consumption. Recent reports indicate a very high correlation between the growing fast food culture and lung cancer incidence. Benzo[a]pyrene (B[a]P) is a potent carcinogen abundantly present in grilled and deep-fried food and in tobacco smoke. Our previous studies have proved the efficacy of curcumin in curbing B[a]P-induced lung carcinogenesis. However, the poor pharmacokinetic profile of the compound considerably hampers its potential as an effective chemopreventive. This study was intended to evaluate whether encapsulation of curcumin in chitosan nanoparticles can improve the cellular uptake and prolong the tissue retention of curcumin yielding better chemoprevention. The curcumin-loaded chitosan nanoparticles (chitosan nanocurcumin) exhibited a size of 170-200 nm in transmission electron microscopy. In vitro drug release studies showed sustained release of curcumin over a period of approximately 180 hours and excellent intracellular uptake and cytotoxicity in lung cancer cells. Bioavailability studies using healthy Swiss albino mice demonstrated drastic enhancement in lung localization of chitosan nanocurcumin compared with free curcumin. Toxicologic evaluation using chronic toxicity model in Swiss albino mice confirmed the pharmacologic safety of the formulation. Moreover, the formulation, even at a dose equivalent to one fourth that of free curcumin, exhibits better efficacy in reducing tumor incidence and multiplicity than free curcumin, thereby hampering development of B[a]P-induced lung adenocarcinomas in Swiss albino mice. Hence, our study underscores the supremacy of the formulation over free curcumin and establishes it as a potential chemopreventive and oral supplement against environmental carcinogenesis.

In silico screening for identification of fatty acid synthase inhibitors and evaluation of their antiproliferative activity using human cancer cell lines.

De novo lipogenesis (DNL) by upregulation of fatty acid synthase (FASN) is an important metabolic alteration of cancer cells. FASN is over-expressed in several cancers and is often associated with a high risk of recurrence and poor prognosis. Differential expression of FASN in cancer cells and their normal counterparts leads to the impression that FASN can be an attractive druggable target in cancer therapy. Present study focuses on identification of inhibitors against FASN ketoacyl synthase (KS) domain from Asinex Biodesign compound database using in silico tools. Virtual screening resulted in the identification of two hit compounds BDD27845077 and BDD27845082 with a common core structure. Molecular Docking studies showed that BDD27845077 and BDD27845082 bind at the substrate entry channel of KS domain with GScore -12.03 kcal/mol and -12.29 kcal/mol respectively. Molecular dynamics (MD) simulation of the protein-ligand complexes shows the binding stability of ligands with FASN-KS. In vitro validation of BDD27845082 demonstrated that the compound possesses antiproliferative activity in a panel of human cancer cell lines including MDA-MB-231 (breast cancer), HCT-116 (colon cancer) and HeLa (cervical cancer) with maximum sensitivity against HCT-116 (IC 50 = 25 µM). The study put forward two lead compounds against FASN with favorable pharmacokinetic profile as indicated by virtual screening tools for the development of cancer chemotherapeutics.

Heteronemin, a marine natural product, sensitizes acute myeloid leukemia cells towards cytarabine chemotherapy by regulating farnesylation of Ras.

Cytarabine is a conventionally used chemotherapeutic agent for treating acute myeloid leukemia (AML). However, chemoresistance, toxic side-effects and poor patient survival rates retard the efficacy of its performance. The current study deals with the chemosensitization of AML cells using heteronemin, a marine natural product towards cytarabine chemotherapy. Heteronemin could effectively sensitize HL-60 cells towards sub-toxic concentration of cytarabine resulting in synergistic toxicity as demonstrated by MTT assay and [3H] thymidine incorporation studies, while being safe towards healthy blood cells. Flow cytometry for Annexin-V/PI and immunoblotting for caspase cleavage proved that the combination induces enhancement in apoptosis. Heteronemin being a farnesyl transferase inhibitor (FTI) suppressed cytarabine-induced, farnesyl transferase-mediated activation of Ras, as assessed by Ras pull-down assay. Upon pre-treating cells with a commercial FTI, L-744,832, the synergism was completely lost in the combination, confirming the farnesyl transferase inhibitory activity of heteronemin as assessed by thymidine incorporation assay. Heteronemin effectively down-regulated cytarabine-induced activation of MAPK, AP-1, NF-κB and c-myc, the down-stream targets of Ras signaling, which again validated the role of Ras in regulating the synergism. Hence we believe that the efficacy of cytarabine chemotherapy can be improved to a significant extent by combining sub-toxic concentrations of cytarabine and heteronemin.

Folic acid conjugation improves the bioavailability and chemosensitizing efficacy of curcumin-encapsulated PLGA-PEG nanoparticles towards paclitaxel chemotherapy.

Nanoencapsulation has emerged as a novel strategy to enhance the pharmacokinetic and therapeutic potential of conventional drugs. Recent studies from our lab have established the efficacy of curcumin in sensitizing cervical cancer cells and breast cancer cells towards paclitaxel and 5-FU chemotherapy respectively. Factors that hinder the clinical use of curcumin as a sensitizer or therapeutic agent include its poor bioavailability and retention time. Earlier reports of improvement in bioavailability and retention of drugs upon nanoencapsulation have motivated us in developing various nanoformulations of curcumin, which were found to exhibit significant enhancement in bioavailability and retention time as assessed by our previous in vitro studies. Among the various formulations tested, curcumin-entrapped in PLGA-PEG nanoparticles conjugated to folic acid (PPF-curcumin) displayed maximum cell death. In the present study, we have demonstrated the efficacy of this formulation in augmenting the bioavailability and retention time of curcumin, in vivo, in Swiss albino mice. Further, the acute and chronic toxicity studies proved that the formulation is pharmacologically safe. We have also evaluated its potential in chemosensitizing cervical cancer cells to paclitaxel and have verified the results using cervical cancer xenograft model in NOD-SCID mice. Folic acid conjugation significantly enhanced the efficacy of curcumin in down-regulating various survival signals induced by paclitaxel in cervical cancer cells and have considerably improved its potential in inhibiting the tumor growth of cervical cancer xenografts. The non-toxic nature coupled with improved chemosensitization potential makes PPF-curcumin a promising candidate formulation for clinical trials.

Radiofrequency ablation of drug-resistant cancer cells using molecularly targeted carboxyl-functionalized biodegradable graphene.

Under ultralow radiofrequency (RF) power, transferrin-conjugated graphene nanoparticles can thermally ablate drug- or radiation-resistant cancer cells very effectively. The results suggest that graphene-based RF hyperthermia can be an efficient method to manage drug-/radiation-resistant cancers.

Rationally designed aberrant kinase-targeted endogenous protein nanomedicine against oncogene mutated/amplified refractory chronic myeloid leukemia.

Deregulated protein kinases play a very critical role in tumorigenesis, metastasis, and drug resistance of cancer. Although molecularly targeted small molecule kinase inhibitors (SMI) are effective against many types of cancer, point mutations in the kinase domain impart drug resistance, a major challenge in the clinic. A classic example is chronic myeloid leukemia (CML) caused by BCR-ABL fusion protein, wherein a BCR-ABL kinase inhibitor, imatinib (IM), was highly successful in the early chronic phase of the disease, but failed in the advanced stages due to amplification of oncogene or point mutations in the drug-binding site of kinase domain. Here, by identifying critical molecular pathways responsible for the drug-resistance in refractory CML patient samples and a model cell line, we have rationally designed an endogenous protein nanomedicine targeted to both cell surface receptors and aberrantly activated secondary kinase in the oncogenic network. Molecular diagnosis revealed that, in addition to point mutations and amplification of oncogenic BCR-ABL kinase, relapsed/refractory patients exhibited significant activation of STAT5 signaling with correlative overexpression of transferrin receptors (TfR) on the cell membrane. Accordingly, we have developed a human serum albumin (HSA) based nanomedicine, loaded with STAT5 inhibitor (sorafenib), and surface conjugated the same with holo-transferrin (Tf) ligands for TfR specific delivery. This dual-targeted "transferrin conjugated albumin bound sorafenib" nanomedicine (Tf-nAlb-Soraf), prepared using aqueous nanoprecipitation method, displayed uniform spherical morphology with average size of ∼150 nm and drug encapsulation efficiency of ∼74%. TfR specific uptake and enhanced antileukemic activity of the nanomedicine was found maximum in the most drug resistant patient sample having the highest level of STAT5 and TfR expression, thereby confirming the accuracy of our rational design and potential of dual-targeting approach. The nanomedicine induced downregulation of key survival pathways such as pSTAT5 and antiapoptotic protein MCL-1 was demonstrated using immunoblotting. This study reveals that, by implementing molecular diagnosis, personalized nanomedicines can be rationally designed and nanoengineered by imparting therapeutic functionality to endogenous proteins to overcome clinically important challenges like molecular drug resistance.

Development and haematotoxicological evaluation of doped hydroxyapatite based multimodal nanocontrast agent for near-infrared, magnetic resonance and X-ray contrast imaging.

Multimodal molecular imaging provides both anatomical and molecular information, aiding early stage detection and better treatment planning of diseased conditions. Here, we report development and nanotoxicity evaluation of a novel hydroxyapatite nanoparticle (nHAp) based multimodal contrast agent for combined near-infrared (NIR), MR and X-ray imaging. Under optimised wet-chemical conditions, we achieved simultaneous doping of nHAp (size ∼50 nm) with indocyanine green and Gd(3+) contributing to NIR contrast (∼750-850 nm), paramagnetic behaviour and X-ray absorption suitable for NIR, MR and X-ray contrast imaging, respectively. Haematocompatibility studies using stem cell viability, haemolysis, platelet activation, platelet aggregation and coagulation time analysis indicated excellent compatibility of doped nHAp (D-nHAp). Further, the immunogenic function studies using human lymphocytes (in vitro) showed that D-nHAp caused no adverse effects. Collectively, our studies suggest that D-nHAp with excellent biocompatibility and multifunctional properties is a promising nanocontrast agent for combined NIR, MR and X-ray imaging applications.