Hyperubiquitylation of DNA helicase RECQL4 by E3 ligase MITOL prevents mitochondrial entry and potentiates mitophagy.

Mutations in the DNA helicase RECQL4 lead to Rothmund-Thomson syndrome (RTS), a disorder characterized by mitochondrial dysfunctions, premature aging, and genomic instability. However, the mechanisms by which these mutations lead to pathology are unclear. Here we report that RECQL4 is ubiquitylated by a mitochondrial E3 ligase, MITOL, at two lysine residues (K1101, K1154) via K6 linkage. This ubiquitylation hampers the interaction of RECQL4 with mitochondrial importer Tom20, thereby restricting its own entry into mitochondria. We show the RECQL4 2K mutant (where both K1101 and K1154 are mutated) has increased entry into mitochondria and demonstrates enhanced mitochondrial DNA (mtDNA) replication. We observed that the three tested RTS patient mutants were unable to enter the mitochondria and showed decreased mtDNA replication. Furthermore, we found that RECQL4 in RTS patient mutants are hyperubiquitylated by MITOL and form insoluble aggregate-like structures on the outer mitochondrial surface. However, depletion of MITOL allows RECQL4 expressed in these RTS mutants to enter mitochondria and rescue mtDNA replication. Finally, we show increased accumulation of hyperubiquitylated RECQL4 outside the mitochondria leads to the cells being potentiated to increased mitophagy. Hence, we conclude regulating the turnover of RECQL4 by MITOL may have a therapeutic effect in patients with RTS.

MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria.

Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated PolγA may have beneficial consequences for certain PEO patients.

CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.

Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival and thereby could be used as a prognostic biomarker. This article has an associated First Person interview with the first author of the paper.

Bile Acid Tethered Docetaxel-Based Nanomicelles Mitigate Tumor Progression through Epigenetic Changes.

In this study, we describe the engineering of sub-100 nm nanomicelles (DTX-PC NMs) derived from phosphocholine derivative of docetaxel (DTX)-conjugated lithocholic acid (DTX-PC) and poly(ethylene glycol)-tethered lithocholic acid. Administration of DTX-PC NMs decelerate tumor progression and increase the mice survivability compared to Taxotere (DTX-TS), the FDA-approved formulation of DTX. Unlike DTX-TS, DTX-PC NMs do not cause any systemic toxicity and slow the decay rate of plasma DTX concentration in rodents and non-rodent species including non-human primates. We further demonstrate that DTX-PC NMs target demethylation of CpG islands of Sparcl1 (a tumor suppressor gene) by suppressing DNA methyltransferase activity and increase the expression of Sparcl1 that leads to tumor regression. Therefore, this unique system has the potential to improve the quality of life in cancer patients and can be translated as a next-generation chemotherapeutic.

Abrogation of FBW7α-dependent p53 degradation enhances p53's function as a tumor suppressor.

The gene encoding the tumor suppressor p53 is mutated in most cancers. p53 expression is known to be tightly controlled by several E3 ligases. Here, we show that F-box and WD repeat domain-containing 7α (FBW7α), the substrate-recognition component of the SCFFBW7 multiprotein E3 ligase complex, targets both WT and tumor-derived mutants of p53 for proteasomal degradation in multiple human cancer cell lines (HCT116 and U2OS). We found that lack of FBW7α stabilizes p53 levels, thereby increasing its half-life. p53 ubiquitylation and subsequent degradation require the F-box and the C-terminal WD40 repeats in FBW7α. The polyubiquitylation of p53 occurred via Lys-48 linkage and involved phosphorylation on p53 at Ser-33 and Ser-37 by glycogen synthase kinase 3ß (GSK3ß) and DNA-dependent protein kinase (DNA-PK), respectively. These phosphorylation events created a phosphodegron that enhanced p53 binding to FBW7α, allowing for the attachment of polyubiquitin moieties at Lys-132 in p53. FBW7α-dependent p53 polyubiquitylation apparently occurred during and immediately after DNA double-strand breaks induced by either doxorubicin or ionizing radiation. Accordingly, in cells lacking FBW7α, p53 induction was enhanced after DNA damage. Phosphodegron-mediated polyubiquitylation of p53 on Lys-132 had functional consequences, with cells in which FBW7α-mediated p53 degradation was abrogated exhibiting enhancement of their tumorigenic potential. We conclude that p53, which previously has been reported to transactivate FBW7, is also targeted by the same E3 ligase for degradation, suggesting the presence of a regulatory feedback loop that controls p53 levels and functions during DNA damage.

Mitochondrial functions of RECQL4 are required for the prevention of aerobic glycolysis-dependent cell invasion.

Germline mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome, which is characterized by a predisposition to cancer. RECQL4 localizes to the mitochondria, where it acts as an accessory factor during mitochondrial DNA replication. To understand the specific mitochondrial functions of RECQL4, we created isogenic cell lines, in which the mitochondrial localization of the helicase was either retained or abolished. The mitochondrial integrity was affected due to the absence of RECQL4 in mitochondria, leading to a decrease in F1F0-ATP synthase activity. In cells where RECQL4 does not localize to mitochondria, the membrane potential was decreased, whereas ROS levels increased due to the presence of high levels of catalytically inactive SOD2. Inactive SOD2 accumulated owing to diminished SIRT3 activity. Lack of the mitochondrial functions of RECQL4 led to aerobic glycolysis that, in turn, led to an increased invasive capability within these cells. Together, this study demonstrates for the first time that, owing to its mitochondrial functions, the accessory mitochondrial replication helicase RECQL4 prevents the invasive step in the neoplastic transformation process.

Ubiquitin-dependent recruitment of the Bloom syndrome helicase upon replication stress is required to suppress homologous recombination.

Limiting the levels of homologous recombination (HR) that occur at sites of DNA damage is a major role of BLM helicase. However, very little is known about the mechanisms dictating its relocalization to these sites. Here, we demonstrate that the ubiquitin/SUMO-dependent DNA damage response (UbS-DDR), controlled by the E3 ligases RNF8/RNF168, triggers BLM recruitment to sites of replication fork stalling via ubiquitylation in the N-terminal region of BLM and subsequent BLM binding to the ubiquitin-interacting motifs of RAP80. Furthermore, we show that this mechanism of BLM relocalization is essential for BLM's ability to suppress excessive/uncontrolled HR at stalled replication forks. Unexpectedly, we also uncovered a requirement for RNF8-dependent ubiquitylation of BLM and PML for maintaining the integrity of PML-associated nuclear bodies and as a consequence the localization of BLM to these structures. Lastly, we identified a novel role for RAP80 in preventing proteasomal degradation of BLM in unstressed cells. Taken together, these data highlight an important biochemical link between the UbS-DDR and BLM-dependent pathways involved in maintaining genome stability.

Tethering of Chemotherapeutic Drug/Imaging Agent to Bile Acid-Phospholipid Increases the Efficacy and Bioavailability with Reduced Hepatotoxicity.

Weakly basic drugs display poor solubility and tend to precipitate in the stomach's acidic environment causing reduced oral bioavailability. Tracing of these orally delivered therapeutic agents using molecular probes is challenged due to their poor absorption in the gastrointestinal tract (GIT). Therefore, we designed a gastric pH stable bile acid derived amphiphile where Tamoxifen (as a model anticancer drug) is conjugated to lithocholic acid derived phospholipid (LCA-Tam-PC). In vitro studies suggested the selective nature of LCA-Tam-PC for cancer cells over normal cells as compared to the parent drug. Fluorescent labeled version of the conjugate (LCA-Tam-NBD-PC) displayed an increased intracellular uptake compared to Tamoxifen. We then investigated the antitumor potential, toxicity, and median survival in 4T1 tumor bearing BALB/c mice upon LCA-Tam-PC treatment. Our studies confirmed a significant reduction in the tumor volume, tumor weight, and reduced hepatotoxicity with a significant increase in median survival on LCA-Tam-PC treatment as compared to the parent drug. Pharmacokinetic and biodistribution studies using LCA-Tam-NBD-PC witnessed the enhanced gut absorption, blood circulation, and tumor site accumulation of phospholipid-drug conjugate leading to improved antitumor activity. Therefore, our studies revealed that conjugation of chemotherapeutic/imaging agents to bile acid phospholipid can provide a new platform for oral delivery and tracing of chemotherapeutic drugs.

Molecular Self-Assembly of Bile Acid-Phospholipids Controls the Delivery of Doxorubicin and Mice Survivability.

Lipid composition in general determines the drug encapsulation efficacy and release kinetics from liposomes that impact the clinical outcomes of cancer therapy. We synthesized three bile acid phospholipids by conjugating the phosphocholine headgroup to the 3'-hydroxyl group of benzylated lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA); and investigated the impact of membrane rigidity on drug encapsulation efficacy, drug release kinetics, anticancer effects, and mice survival. Liposomes with a hydrodynamic diameter of 100-110 nm were subsequently developed using these phospholipids. Fluorescence-probe based quantification revealed a more fluidic nature of DCA-PC- and CA-PC-derived liposomes, whereas the LCA-PC-derived ones are rigid in nature. Doxorubicin encapsulation studies showed ∼75% encapsulation and ∼38% entrapment efficacy of doxorubicin using more fluidic DCA-PC and CA-PC derived liposomes as compared to ∼58% encapsulation and ∼18% entrapment efficacy in the case of LCA-PC derived liposomes. In vivo anticancer studies in the murine model confirmed that doxorubicin entrapped CA-PC liposomes compromise mice survival, whereas rigid drug entrapped LCA-PC-derived-liposomes increased mice survival with ∼2-fold decrease in tumor volume. Pharmacokinetic and biodistribution studies revealed an ∼1.5-fold increase in plasma drug concentration and an ∼4.0-fold rise in tumor accumulation of doxorubicin on treatment with drug entrapped LCA-PC liposomes as compared to doxorubicin alone. In summary, this study presents the impact of bile acid derived liposomes with different rigidities on drug delivery and mice survivability.

Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function.

A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions.

Enhancement of c-Myc degradation by BLM helicase leads to delayed tumor initiation.

The spectrum of tumors that arise owing to the overexpression of c-Myc and loss of BLM is very similar. Hence, it was hypothesized that the presence of BLM negatively regulates c-Myc functions. By using multiple isogenic cell lines, we observed that the decrease of endogenous c-Myc levels that occurs in the presence of BLM is reversed when the cells are treated with proteasome inhibitors, indicating that BLM enhances c-Myc turnover. Whereas the N-terminal region of BLM interacts with c-Myc, the rest of the helicase interacts with the c-Myc E3 ligase Fbw7. The two BLM domains act as 'clamp and/or adaptor', enhancing the binding of c-Myc to Fbw7. BLM promotes Fbw7-dependent K48-linked c-Myc ubiquitylation and its subsequent degradation in a helicase-independent manner. A subset of BLM-regulated genes that are also targets of c-Myc were determined and validated at both RNA and protein levels. To obtain an in vivo validation of the effect of BLM on c-Myc-mediated tumor initiation, isogenic cells from colon cancer cells that either do or do not express BLM had been manipulated to block c-Myc expression in a controlled manner. By using these cell lines, the metastatic potential and rate of initiation of tumors in nude mice were determined. The presence of BLM decreases c-Myc-mediated invasiveness and delays tumor initiation in a mouse xenograft model. Consequently, in tumors that express BLM but not c-Myc, we observed a decreased ratio of proliferation to apoptosis together with a suppressed expression of the angiogenesis marker CD31. Hence, partly owing to its regulation of c-Myc stability, BLM acts as a 'caretaker tumor suppressor'.

RECQL4 and p53 potentiate the activity of polymerase γ and maintain the integrity of the human mitochondrial genome.

UNLABELLED: Germline mutations in RECQL4 and p53 lead to cancer predisposition syndromes, Rothmund-Thomson syndrome (RTS) and Li-Fraumeni syndrome (LFS), respectively. RECQL4 is essential for the transport of p53 to the mitochondria under unstressed conditions. Here, we show that both RECQL4 and p53 interact with mitochondrial polymerase (PolγA/B2) and regulate its binding to the mitochondrial DNA (mtDNA) control region (D-loop). Both RECQL4 and p53 bind to the exonuclease and polymerase domains of PolγA. Kinetic constants for interactions between PolγA-RECQL4, PolγA-p53 and PolγB-p53 indicate that RECQL4 and p53 are accessory factors for PolγA-PolγB and PolγA-DNA interactions. RECQL4 enhances the binding of PolγA to DNA, thereby potentiating the exonuclease and polymerization activities of PolγA/B2. To investigate whether lack of RECQL4 and p53 results in increased mitochondrial genome instability, resequencing of the entire mitochondrial genome was undertaken from multiple RTS and LFS patient fibroblasts. We found multiple somatic mutations and polymorphisms in both RTS and LFS patient cells. A significant number of mutations and polymorphisms were common between RTS and LFS patients. These changes are associated with either aging and/or cancer, thereby indicating that the phenotypes associated with these syndromes may be due to deregulation of mitochondrial genome stability caused by the lack of RECQL4 and p53. SUMMARY: The biochemical mechanisms by which RECQL4 and p53 affect mtDNA replication have been elucidated. Resequencing of RTS and LFS patients' mitochondrial genome reveals common mutations indicating similar mechanisms of regulation by RECQL4 and p53.

Inositol pyrophosphate synthesis by inositol hexakisphosphate kinase 1 is required for homologous recombination repair.

Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP(7)), are water-soluble inositol phosphates that contain high energy diphosphate moieties on the inositol ring. Inositol hexakisphosphate kinase 1 (IP6K1) participates in inositol pyrophosphate synthesis, converting inositol hexakisphosphate (IP(6)) to IP(7). In the present study, we show that mouse embryonic fibroblasts (MEFs) lacking IP6K1 exhibit impaired DNA damage repair via homologous recombination (HR). IP6K1 knock-out MEFs show decreased viability and reduced recovery after induction of DNA damage by the replication stress inducer, hydroxyurea, or the radiomimetic antibiotic, neocarzinostatin. Cells lacking IP6K1 arrest after genotoxic stress, and markers associated with DNA repair are recruited to DNA damage sites, indicating that HR repair is initiated in these cells. However, repair does not proceed to completion because these markers persist as nuclear foci long after drug removal. A fraction of IP6K1-deficient MEFs continues to proliferate despite the persistence of DNA damage, rendering the cells more susceptible to chromosomal aberrations. Expression of catalytically active but not inactive IP6K1 can restore the repair process in knock-out MEFs, implying that inositol pyrophosphates are required for HR-mediated repair. Our study therefore highlights inositol pyrophosphates as novel small molecule regulators of HR signaling in mammals.

RECQL4 is essential for the transport of p53 to mitochondria in normal human cells in the absence of exogenous stress.

Mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome (RTS). A subset of RTS patients is predisposed to cancer and is sensitive to DNA damaging agents. The enhanced sensitivity of cells from RTS patients correlates with the accumulation of transcriptionally active nuclear p53. We found that in untreated normal human cells these two nuclear proteins, p53 and RECQL4, instead colocalize in the mitochondrial nucleoids. RECQL4 accumulates in mitochondria in all phases of the cell cycle except S phase and physically interacts with p53 only in the absence of DNA damage. p53-RECQL4 binding leads to the masking of the nuclear localization signal of p53. The N-terminal 84 amino acids of RECQL4 contain a mitochondrial localization signal, which causes the localization of RECQL4-p53 complex to the mitochondria. RECQL4-p53 interaction is disrupted after stress, allowing p53 translocation to the nucleus. In untreated normal cells RECQL4 optimizes de novo replication of mtDNA, which is consequently decreased in fibroblasts from RTS patients. Wild-type RECQL4-complemented RTS cells show relocalization of both RECQL4 and p53 to the mitochondria, loss of p53 activation, restoration of de novo mtDNA replication and resistance to different types of DNA damage. In cells expressing Δ84 RECQL4, which cannot translocate to mitochondria, all the above functions are compromised. The recruitment of p53 to the sites of de novo mtDNA replication is also regulated by RECQL4. Thus these findings elucidate the mechanism by which p53 is regulated by RECQL4 in unstressed normal cells and also delineates the mitochondrial functions of the helicase.

Mammalian proapoptotic factor ChaC1 and its homologues function as γ-glutamyl cyclotransferases acting specifically on glutathione.

ChaC1 is a mammalian proapoptic protein of unknown function induced during endoplasmic reticulum stress. We show using in vivo studies and novel in vitro assays that the ChaC family of proteins function as γ-glutamyl cyclotransferases acting specifically to degrade glutathione but not other γ-glutamyl peptides. The overexpression of these proteins (but not the catalytically dead E>Q mutants) led to glutathione depletion and enhanced apoptosis in yeast. The ChaC family is conversed across all phyla and represents a new pathway for glutathione degradation in living cells, and the first cytosolic pathway for glutathione degradation in mammalian cells.

Small molecules that disrupt RAD54-BLM interaction hamper tumor proliferation in colon cancer chemoresistance models.

RAD54 and BLM helicase play pivotal roles during homologous recombination repair (HRR) to ensure genome maintenance. BLM amino acids (aa 181-212) interact with RAD54 and enhance its chromatin remodeling activity. Functionally, this interaction heightens HRR, leading to a decrease in residual DNA damage in colon cancer cells. This contributes to chemoresistance in colon cancer cells against cisplatin, camptothecin, and oxaliplatin, eventually promoting tumorigenesis in preclinical colon cancer mouse models. ChIP-Seq analysis and validation revealed increased BLM and RAD54 corecruitment on the MRP2 promoter in camptothecin-resistant colon cancer cells, leading to BLM-dependent enhancement of RAD54-mediated chromatin remodeling. We screened the Prestwick small-molecule library, with the intent to revert camptothecin- and oxaliplatin-induced chemoresistance by disrupting the RAD54-BLM interaction. Three FDA/European Medicines Agency-approved candidates were identified that could disrupt this interaction. These drugs bound to RAD54, altered its conformation, and abrogated RAD54-BLM-dependent chromatin remodeling on G5E4 and MRP2 arrays. Notably, the small molecules also reduced HRR efficiency in resistant lines, diminished anchorage-independent growth, and hampered the proliferation of tumors generated using camptothecin- and oxaliplatin-resistant colon cancer cells in both xenograft and syngeneic mouse models in BLM-dependent manner. Therefore, the 3 identified small molecules can serve as possible viable candidates for adjunct therapy in colon cancer treatment.

Engineered nanomicelles inhibit the tumour progression via abrogating the prostaglandin-mediated immunosuppression.

Cancer treatment is challenged due to immunosuppressive inflammatory tumour microenvironment (TME) caused by infiltration of tumour-promoting and inhibition of tumour-inhibiting immune cells. Here, we report the engineering of chimeric nanomicelles (NMs) targeting the cell proliferation using docetaxel (DTX) and inflammation using dexamethasone (DEX) that alters the immunosuppressive TME. We show that a combination of phospholipid-DTX conjugate and PEGylated-lipid-DEX conjugate can self-assemble to form sub-100 nm chimeric NMs (DTX-DEX NMs). Anti-cancer activities against syngeneic and xenograft mouse models showed that the DTX-DEX NMs are more effective in tumour regression, enhance the survival of mice over other treatment modes, and alter the tumour stroma. DTX-DEX NMs cause a significant reduction in myeloid-derived suppressor cells, alter the polarization of macrophages, and enhance the accumulation of cytotoxic CD4+ and CD8+ T cells in tumour tissues, along with alterations in cytokine expression. We further demonstrated that these DTX-DEX NMs inhibit the synthesis of prostaglandins, especially PGE2, by targeting the cyclooxygenase 2 that is partly responsible for immunosuppressive TME. Therefore, this study presents, for the first time, the engineering of lithocholic acid-derived chimeric NMs that affect the prostaglandin pathway, alter the TME, and mitigate tumour progression with enhanced mice survival.

Design, synthesis, and mechanistic investigations of bile acid-tamoxifen conjugates for breast cancer therapy.

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA). These bile acid-tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The in vitro anticancer activities of these bile acid-tamoxifen conjugates show that the free amine headgroup based cholic acid-tamoxifen conjugate (CA-Tam3-Am) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid-tamoxifen conjugates. The cholic acid-tamoxifen conjugate (CA-Tam3-Am) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor -ve breast cancer cell lines. The enhanced anticancer activity of CA-Tam3-Am is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G0 phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome c, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER -ve cells. In vivo studies in the 4T1 tumor model showed that CA-Tam3-Am is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid-drug conjugates.

Transglutaminase-Polyethyleneimine Nanoflowers Mediated Cellular Delivery of Anti-miR-210 for Effective Glioblastoma Therapy.

Glioblastoma (GBM) is a deadly tumor of the central nervous system (CNS) having a dismal prognosis. miRNA-based therapeutics hold immense potential for GBM therapy; however, its delivery remains a daunting challenge. MicroRNA-210 has been established as a critical oncomiR in GBM. Our group has developed novel, PEI-functionalized transglutaminase-based nanoflowers (TGNFs, ∼61 nm in diameter) for the efficient delivery of anti-miR-210 to glioblastoma cells in vitro. TGNFs show low cytotoxicity to normal human fibroblasts, do not affect the liver and kidney health of CD1 mice, and offer >95% anti-miR encapsulation efficiency, serum stability, and protection against polyanion moieties. Their synthesis is cost-effective and does not involve the application of harsh chemicals. TGNFs successfully delivered anti-miR-210 to glioblastoma cells, decreasing cellular proliferation and migration and increasing apoptosis. Overall, this research highlights the potential of TGNFs as delivery agents in miRNA inhibition therapy and encourages further preclinical studies to explore the potential of miR-210 as a therapeutic target in GBM and various other cancers where the oncogenic role of miR-210 has been well-established.