A designer bow-tie combination therapeutic platform: An approach to resistant cancer treatment by simultaneous delivery of cytotoxic and anti-inflammatory agents and radiation.

Multimodal therapies are used to treat advanced cancers including castration-resistant prostate cancer to manage the biological characteristics of the tumors like inflammation, bone metastases, and participation of metabolically altered cancer stem cells (CSCs) that have integral roles in disease dissemination and progression. We developed a multifunctional polymer-based self-assembled technology to deliver a predefined stoichiometric combination of a chemotherapy and an anti-inflammatory agent in a stimuli responsive manner, to complement and improve the currently established treatment methods of prostate cancer. We combined clinically applicable fractionated radiation therapy (XRT) to further sensitize the activity of this targeted multifunctional platform towards prostate-specific membrane antigen (PSMA) expressing advanced prostate cancer. After irradiation, our PSMA-targeted self-assembly system could modulate the mitochondrial metabolism, cellular respiration and the overall radiation-induced DNA damage process. We report the synthesis of this advanced multifunctional platform and describe its unique properties that allow the ability to load multiple FDA approved drugs with a predefined stoichiometric ratio for targeted co-delivery of chemotherapeutics and anti-inflammatory agents. The efficacy of this platform was demonstrated using several in vitro and in vivo studies, in a unique bilateral PSMA expressing prostate cancer tumor model, and in patient derived CSCs.

SET contributes to the epithelial-mesenchymal transition of pancreatic cancer.

Pancreatic cancer has a devastating prognosis due to 80-90% of diagnostic cases occurring when metastasis has already presented. Activation of the epithelial-mesenchymal transition (EMT) is a prerequisite for metastasis because it allows for the dissemination of tumor cells to blood stream and secondary organs. Here, we sought to determine the role of SET oncoprotein, an endogenous inhibitor of PP2A, in EMT and pancreatic tumor progression. Among the two major isoforms of SET (isoform 1 and isoform 2), higher protein levels of SET isoform 2 were identified in aggressive pancreatic cancer cell lines. Overexpressing SET isoform 2, and to a lesser extent SET isoform 1, in epithelial cell lines promoted EMT-like features by inducing mesenchymal characteristics and promoting cellular proliferation, migration, invasion, and colony formation. Consistently, knockdown of SET isoforms in the mesenchymal cell line partially resisted these characteristics and promoted epithelial features. SET-induced EMT was likely facilitated by increased N-cadherin overexpression, decreased PP2A activity and/or increased expression of key EMT-driving transcription factors. Additionally, SET overexpression activated the Rac1/JNK/c-Jun signaling pathway that induced transcriptional activation of N-cadherin expression. In vivo, SET isoform 2 overexpression significantly correlated with increased N-cadherin in human PDAC and to tumor burden and metastatic ability in an orthotopic mouse tumor model. These findings identify a new role for SET in cancer and have implications for the design and targeting of SET for intervening pancreatic tumor progression.

miR-202 Diminishes TGFß Receptors and Attenuates TGFß1-Induced EMT in Pancreatic Cancer.

Previous studies in our laboratory identified that 3-deazaneplanocin A (DZNep), a carbocyclic adenosine analog and histone methyl transferase inhibitor, suppresses TGFß-induced epithelial-to-mesenchymal (EMT) characteristics. In addition, DZNep epigenetically reprograms miRNAs to regulate endogenous TGFß1 levels via miR-663/4787-mediated RNA interference (Mol Cancer Res. 2016 Sep 13. pii: molcanres.0083.2016) (1). Although DZNep also attenuates exogenous TGFß-induced EMT response, the mechanism of this inhibition was unclear. Here, DZNep induced miR-202-5p to target both TGFß receptors, TGFBR1 and TGFBR2, for RNA interference and thereby contributes to the suppression of exogenous TGFß-induced EMT in pancreatic cancer cells. Lentiviral overexpression of miR-202 significantly reduced the protein levels of both TGFß receptors and suppressed TGFß signaling and EMT phenotypic characteristics of cultured parenchymal pancreatic cancer cells. Consistently, transfection of anti-miRNAs against miR-202-5p resulted in increased TGFBR1 and TGFBR2 protein expressions and induced EMT characteristics in these cells. In stellate pancreatic cells, miR-202 overexpression slowed growth as well as reduced stromal extracellular membrane matrix protein expression. In orthotopic pancreatic cancer mouse models, both immunodeficient and immunocompetent, miR-202 reduced tumor burden and metastasis. Together, these findings demonstrate an alternative mechanism of DZNep in suppressing TGFß signaling at the receptor level and uncover the EMT-suppressing role of miR-202 in pancreatic cancer.Implications: These findings support the possibility of combining small molecule-based (e.g., DZNep analogs) or large molecule-based (e.g., miRNAs) epigenetic modifiers with conventional nucleoside analogs (e.g., gemcitabine, capecitabine) to improve the antimetastatic potential of current pancreatic cancer therapy. Mol Cancer Res; 15(8); 1029-39. ©2017 AACR.

A Prodrug of Two Approved Drugs, Cisplatin and Chlorambucil, for Chemo War Against Cancer.

Cancer cells maintain normal mitochondrial glutathione as one of the defense mechanisms to inhibit mitochondrial membrane polarization and hence apoptosis. A combinational therapeutic modality Platin-Cbl, a prodrug of FDA-approved chemotherapeutic agents, cisplatin and chlorambucil (Cbl), was synthesized and characterized to explore the potential of this compound to initiate chemo war on cancer cells using the active drugs, cisplatin and Cbl, when delivered to the cellular power house mitochondrion using a targeted nanoparticle designed to get associated with this organelle. Platin-Cbl demonstrated significantly high cytotoxic activity across a number of tumor cell lines as well as in a cisplatin-resistant cancer cell line compared with cisplatin or its mixture with Cbl suggesting its unique potency in cisplatin-resistant tumors. A mitochondria-targeted nanoparticle formulation of Platin-Cbl allowed for its efficacious mitochondrial delivery. In vitro studies documented high potency of Platin-Cbl nanoparticle formulations. Cisplatin-resistant cells upon treatment with Platin-Cbl were still able to manage energy production to a certain extent via fatty acid pathway; the advantage of using T-Platin-Cbl-NP is that this nanoparticle treatment causes impairment of all metabolic pathways in cisplatin-resistant cells forcing the cells to undergo efficient apoptosis. This study highlights a combination of several beneficial effects for a cascade of events to overcome resistance associated with single drug therapy. Mol Cancer Ther; 16(4); 625-36. ©2017 AACR.

The Platin-X series: activation, targeting, and delivery.

Anticancer platinum (Pt) complexes have long been considered to be one of the biggest success stories in the history of medicinal inorganic chemistry. Yet there remains the hunt for the "magic bullet" which can satisfy the requirements of an effective chemotherapeutic drug formulation. Pt(iv) complexes are kinetically more inert than the Pt(ii) congeners and offer the opportunity to append additional functional groups/ligands for prodrug activation, tumor targeting, or drug delivery. The ultimate aim of functionalization is to enhance the tumor selective action and attenuate systemic toxicity of the drugs. Moreover, an increase in cellular accumulation to surmount the resistance of the tumor against the drugs is also of paramount importance in drug development and discovery. In this review, we will address the attempts made in our lab to develop Pt(iv) prodrugs that can be activated and delivered using targeted nanotechnology-based delivery platforms.

Nanotechnology inspired tools for mitochondrial dysfunction related diseases.

Mitochondrial dysfunctions are recognized as major factors for various diseases including cancer, cardiovascular diseases, diabetes, neurological disorders, and a group of diseases so called "mitochondrial dysfunction related diseases". One of the major hurdles to gain therapeutic efficiency in diseases where the targets are located in the mitochondria is the accessibility of the targets in this compartmentalized organelle that imposes barriers toward internalization of ions and molecules. Over the time, different tools and techniques were developed to improve therapeutic index for mitochondria acting drugs. Nanotechnology has unfolded as one of the logical and encouraging tools for delivery of therapeutics in controlled and targeted manner simultaneously reducing side effects from drug overdose. Tailor-made nanomedicine based therapeutics can be an excellent tool in the toolbox for diseases associated with mitochondrial dysfunctions. In this review, we present an extensive coverage of possible therapeutic targets in different compartments of mitochondria for cancer, cardiovascular, and mitochondrial dysfunction related diseases.

Unique Use of Alkylation for Chemo-Redox Activity by a Pt(IV) Prodrug.

Resistance towards chemotherapeutics displayed by cancer cells is a significant stumbling block against fruitful cisplatin-based therapy. A unique dual-acting chemotherapeutic modality, Platin-B, a prodrug of cisplatin and pipobroman-mimicking alkylating agent, was constructed to circumvent tumor resistance. Platin-B exhibited a superior cytotoxicity profile in cisplatin-resistant cancer cells. Enhanced activity and the ability to overcome cancer-induced resistance of Platin-B was related to adduct formation with intracellular glutathione, followed by the activity of Platin-B on the mitochondria of cells, along with its conventional nuclear activity. Alkylating moieties present on Platin-B enhanced its cellular and subcellular concentration and protected it from early drug sequestration by biological thiols.

New formulation of old aspirin for better delivery.

For better use of cyclooxygenase dependent anti-inflammatory properties and mitochondrial activities of aspirin, new hydrophobic analogues of aspirin were developed and successfully encapsulated in polymeric nanoparticles (NPs). In vivo anti-inflammatory effects of these NPs using a mouse model demonstrated unique properties of an optimized aspirin analogue to inhibit production of pro-inflammatory and enrichment of anti-inflammatory cytokines.

Evaluation of nanoparticle delivered cisplatin in beagles.

Intracranial neoplasia is a significant cause of morbidity and mortality in both human and veterinary patients, and is difficult to treat with traditional therapeutic methods. Cisplatin is a platinum (Pt)-containing chemotherapeutic agent approved by the Food and Drug Administration; however, substantial limitations exist for its application in canine brain tumor treatment due to the difficulty in crossing the blood-brain barrier (BBB), development of resistance, and toxicity. A modified Pt(iv)-prodrug of cisplatin, Platin-M, was recently shown to be deliverable to the brain via a biocompatible mitochondria-targeted lipophilic polymeric nanoparticle (NP) that carries the drug across the BBB and to the mitochondria. NP mediated controlled release of Platin-M and subsequent reduction of this prodrug to cisplatin allowed cross-links to be formed with the mitochondrial DNA, which have no nucleotide excision repair system, forcing the overactive cancer cells to undergo apoptosis. Here, we report in vitro effects of targeted Platin-M NPs (T-Platin-M-NPs) in canine glioma and glioblastoma cell lines with results indicating that this targeted NP formulation is more effective than cisplatin. In both the cell lines, T-Platin-M-NP was significantly more efficacious compared to carboplatin, another Pt-based chemotherapy, which is used in the settings of recurrent high-grade glioblastoma. Mitochondrial stress analysis indicated that T-Platin-M-NP is more effective in disrupting the mitochondrial bioenergetics in both the cell types. A 14-day distribution study in healthy adult beagles using a single intravenous injection at 0.5 mg kg(-1) (with respect to Platin-M) of T-Platin-M-NPs showed high levels of Pt accumulation in the brain, with negligible amounts in the other analyzed organs. Safety studies in the beagles monitoring physical, hematological, and serum chemistry evaluations were within the normal limits on days 1, 7, and 14 after injection of either 0.5 mg kg(-1) or 2 mg kg(-1) or 2.2 mg kg(-1) (with respect to Platin-M) of T-Platin-M-NPs. At all doses over the 14-day period, no neurotoxicity was observed based upon periodic neurological examinations and cerebrospinal fluid analysis. These studies demonstrated the translational nature of T-Platin-M-NPs for applications in the treatment of brain tumors.

A Nanoparticle Cocktail: Temporal Release of Predefined Drug Combinations.

A single magic bullet is not enough for treatment of metastatic cancers. However, administration of a combination of free drugs can be extremely challenging because of the inability to control the correct choice of dosages and definitive delivery of the effective drug ratio at the target tissue due to the differences in pharmacokinetics and biodistribution of individual drugs. Here we report an engineered biodegradable polymer containing combination therapeutics that can be self-assembled into a controlled release nanoparticle with abilities to deliver multiple therapeutics in a predefined ratio following temporal release patterns. This platform technology can lead to a rationally designed combination therapy.

Targeted nanoparticles in mitochondrial medicine.

Mitochondria, the so-called 'energy factory of cells' not only produce energy but also contribute immensely in cellular mortality management. Mitochondrial dysfunctions result in various diseases including but not limited to cancer, atherosclerosis, and neurodegenerative diseases. In the recent years, targeting mitochondria emerged as an attractive strategy to control mitochondrial dysfunction-related diseases. Despite the desire to direct therapeutics to the mitochondria, the actual task is more difficult due to the highly complex nature of the mitochondria. The potential benefits of integrating nanomaterials with properties such as biodegradability, magnetization, and fluorescence into a single object of nanoscale dimensions can lead to the development of hybrid nanomedical platforms for targeting therapeutics to the mitochondria. Only a handful of nanoparticles based on metal oxides, gold nanoparticles, dendrons, carbon nanotubes, and liposomes were recently engineered to target mitochondria. Most of these materials face tremendous challenges when administered in vivo due to their limited biocompatibility. Biodegradable polymeric nanoparticles emerged as eminent candidates for effective drug delivery. In this review, we highlight the current advancements in the development of biodegradable nanoparticle platforms as effective targeting tools for mitochondrial medicine.

Detouring of cisplatin to access mitochondrial genome for overcoming resistance.

Chemoresistance of cisplatin therapy is related to extensive repair of cisplatin-modified DNA in the nucleus by the nucleotide excision repair (NER). Delivering cisplatin to the mitochondria to attack mitochondrial genome lacking NER machinery can lead to a rationally designed therapy for metastatic, chemoresistant cancers and might overcome the problems associated with conventional cisplatin treatment. An engineered hydrophobic mitochondria-targeted cisplatin prodrug, Platin-M, was constructed using a strain-promoted alkyne-azide cycloaddition chemistry. Efficient delivery of Platin-M using a biocompatible polymeric nanoparticle (NP) based on biodegradable poly(lactic-co-glycolic acid)-block-polyethyleneglycol functionalized with a terminal triphenylphosphonium cation, which has remarkable activity to target mitochondria of cells, resulted in controlled release of cisplatin from Platin-M locally inside the mitochondrial matrix to attack mtDNA and exhibited otherwise-resistant advanced cancer sensitive to cisplatin-based chemotherapy. Identification of an optimized targeted-NP formulation with brain-penetrating properties allowed for delivery of Platin-M inside the mitochondria of neuroblastoma cells resulting in ∼17 times more activity than cisplatin. The remarkable activity of Platin-M and its targeted-NP in cisplatin-resistant cells was correlated with the hyperpolarization of mitochondria in these cells and mitochondrial bioenergetics studies in the resistance cells further supported this hypothesis. This unique dual-targeting approach to controlled mitochondrial delivery of cisplatin in the form of a prodrug to attack the mitochondrial genome lacking NER machinery and in vivo distribution of the delivery vehicle in the brain suggested previously undescribed routes for cisplatin-based therapy.

Copper-free click-chemistry platform to functionalize cisplatin prodrugs.

The ability to rationally design and construct a platform technology to develop new platinum(IV) [Pt(IV)] prodrugs with functionalities for installation of targeting moieties, delivery systems, fluorescent reporters from a single precursor with the ability to release biologically active cisplatin by using well-defined chemistry is critical for discovering new platinum-based therapeutics. With limited numbers of possibilities considering the sensitivity of Pt(IV) centers, we used a strain-promoted azide-alkyne cycloaddition approach to provide a platform, in which new functionalities can easily be installed on cisplatin prodrugs from a single Pt(IV) precursor. The ability of this platform to be incorporated in nanodelivery vehicle and conjugation to fluorescent reporters were also investigated.

Mito-DCA: a mitochondria targeted molecular scaffold for efficacious delivery of metabolic modulator dichloroacetate.

Tumor growth is fueled by the use of glycolysis, which normal cells use only in the scarcity of oxygen. Glycolysis makes tumor cells resistant to normal death processes. Targeting this unique tumor metabolism can provide an alternative strategy to selectively destroy the tumor, leaving normal tissue unharmed. The orphan drug dichloroacetate (DCA) is a mitochondrial kinase inhibitor that has the ability to show such characteristics. However, its molecular form shows poor uptake and bioavailability and limited ability to reach its target mitochondria. Here, we describe a targeted molecular scaffold for construction of a multiple DCA loaded compound, Mito-DCA, with three orders of magnitude enhanced potency and cancer cell specificity compared to DCA. Incorporation of a lipophilic triphenylphosphonium cation through a biodegradable linker in Mito-DCA allowed for mitochondria targeting. Mito-DCA did not show any significant metabolic effects toward normal cells but tumor cells with dysfunctional mitochondria were affected by Mito-DCA, which caused a switch from glycolysis to glucose oxidation and subsequent cell death via apoptosis. Effective delivery of DCA to the mitochondria resulted in significant reduction in lactate levels and played important roles in modulating dendritic cell (DC) phenotype evidenced by secretion of interleukin-12 from DCs upon activation with tumor antigens from Mito-DCA treated cancer cells. Targeting mitochondrial metabolic inhibitors to the mitochondria could lead to induction of an efficient antitumor immune response, thus introducing the concept of combining glycolysis inhibition with immune system to destroy tumor.

The prodrug platin-A: simultaneous release of cisplatin and aspirin.

Cancer-associated inflammation induces tumor progression to the metastatic stage, thus indicating that a chemo-anti-inflammatory strategy is of interest for the management of aggressive cancers. The platinum(IV) prodrug Platin-A was designed to release cisplatin and aspirin to ameliorate the nephrotoxicity and ototoxicity caused by cisplatin. Platin-A exhibited anticancer and anti-inflammatory properties which are better than a combination of cisplatin and aspirin. These findings highlight the advantages of combining anti-inflammatory treatment with chemotherapy when both the drugs are delivered in the form of a single prodrug.

Multiple sensor array of Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+ complexes of a triazole linked imino-phenol based calix[4]arene conjugate for the selective recognition of Asp, Glu, Cys, and His.

The triazole linked o-imino phenol appended calix[4]arene conjugate (L) has been synthesized and characterized. The structure of L has been established based on single crystal XRD. The binding and recognition behavior of conjugate, L toward the transition metal ions, such as Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+), has been demonstrated using fluorescence, absorption and ESI-MS techniques. The in situ prepared complexes of these metal ions, namely, [Mn(2)L], [Fe(2)L], [Co(2)L], [Ni(2)L], [Cu(2)L], and [Zn(2)L] have shown recognition toward Glu, Asp, His and Cys. Hence L provides a multiple sensing molecular tool where the response for the recognition of biologically active amino acids of metalloproteins is elicited by the presence of specific metal ion.

Cd2+ complex of a triazole-based calix[4]arene conjugate as a selective fluorescent chemosensor for Cys.

An N,N-Dimethylamine ethylimino-appended triazole-linked calix[4]arene conjugate, L, has been synthesized and characterized, and its Cd(2+) complex has been isolated and characterized. The structure of [CdL] was established by computational calculation using B3LYP/LANL2DZ. Time-dependent density functional theory calculations were performed to demonstrate the electronic properties of [CdL]. This highly fluorescing [CdL] has been used to recognize Cys selectively among the 20 naturally occurring amino acids. [CdL] exhibits a minimum detection limit of 58 ppb for Cys, with reusability and reversibility being imparted to the system during sensing. Thus, the sensing of Cys was well demonstrated using various techniques, viz., fluorescence, absorption, visual color change, electrospray ionization MS, (1)H NMR, and live cell imaging experiments.