Diverse Farming Systems and their Impact on Macro and Microelement Content of Vegetables & Crops.

BACKGROUND: The present study investigates the effect of conventional and organic farming systems on the nutritional profile of crops. Different crops, namely -millet, sorghum, sesame, mustard, fenugreek, berseem, pea, potato, and onion were cultivated through conventional agriculture in which chemical fertilizers like urea, DAP (Diammonium Phosphate) and pesticides were used and organic farming in which organic fertilizers like seaweed and vermicompost were used. OBJECTIVE: The experimental study was done on a field in north India from 2019 to 2021 in six different seasons, and the nutrient profile of the crops with respect to macroelements (S, K, Na, P, Ca, Mg) and microelements (B, Cu, Fe, Mn, Zn, Al) was compared. METHODS: Macro and microelements were analyzed by Element analyzer and ICP-OES in both types of farming systems. The content of macro, as well as microelements, was found to be significantly higher in all the organically produced crops as compared to the conventionally grown crops. RESULTS: Significant differences were observed in the macroelement content of organic onion (P900 mg/kg, K-2000mg/kg) and organic pea (K 2250 mg/kg) as compared to the content of conventionally grown onion (P-756 mg/kg, K- 1550 mg/kg) and pea (K-2000 mg/kg). Similarly, microelement content in the organic sesame (Fe - 3.12 mg/kg), organic millet (Fe- 2.19 mg/kg), and organic potato (Zn-200 mg/kg) was higher as compared to conventionally grown sesame (Fe 2.05 mg/kg), millet (Fe- 1.56 mg/kg) and potato (Zn 167 mg/kg). CONCLUSION: This investigation concludes that crops with optimum nutritional content can be produced through organic farming with minimum input and maximum production.

Curcumin Suppresses M2 Macrophage-derived Paclitaxel Chemoresistance through Inhibition of PI3K-AKT/STAT3 Signaling.

BACKGROUND: Breast cancer is the leading cancer in women worldwide. The development of chemoresistance that leads to recurrence and mortality remains a major concern. M2-type tumor-associated macrophages (TAMs), present in the breast tumor microenvironment, secrete various cytokines and growth factors that induce chemoresistance. Curcumin, isolated from Curcuma longa, is known to sensitize cancer cells and increase the efficacy of standard chemotherapeutic agents. However, the effect of curcumin on the chemoresistancegenerating ability of M2 TAMs is not known. OBJECTIVE: The study aimed to determine whether curcumin could modulate M2 macrophages and suppress their ability to induce resistance to paclitaxel in breast cancer cells. METHODS: THP-1 cells were differentiated to M2 macrophages using PMA and IL-4/IL-13 in the presence or absence of curcumin in vitro. The effect of the conditioned medium of M2 macrophages on inducing resistance towards paclitaxel in MCF-7 or MDA-MB-231 cells was analyzed by cell proliferation assay, cell cycle analysis, wound healing and transwell migration assays. RT-PCR analysis was used to determine the mRNA expression of anti-inflammatory cytokines in M2 macrophages. The effect of curcumin on TGF-ß, pAKT, and pSTAT3 in M2 macrophages was analyzed by western blotting. RESULTS: Our data revealed that the M2 macrophages polarized in the presence of curcumin lacked the ability to generate chemoresistance to paclitaxel in breast cancer cell lines. Transcriptomic analysis revealed the expression of TGF-ß to be highest amongst M2 macrophage-secreted cytokines. We observed that purified recombinant TGF-ß generated chemoresistance in breast cancer cells. We found that curcumin treatment abrogated the expression of TGF-ß in M2 macrophages and suppressed their ability to induce chemoresistance in breast cancer cells. STITCH analysis showed strong interaction between curcumin and AKT/STAT3 pathway. Mechanistically, curcumin inhibited PI3K/AKT/STAT3 signaling in M2 macrophages. Western blot analysis revealed that M2 TAM CM, but not curcumin-treated macrophage CM, activated COX2/NF-κB in breast cancer cells. CONCLUSION: Our results showed that curcumin reduced the chemoresistance-generating ability of M2 TAMs. The study has revealed a non-cancer cell-autonomous mechanism by which curcumin partly overcomes the chemoresistance of paclitaxel in breast cancer.

Beaver Tail Liver: A Rare Anatomic Variant.

Beaver tail liver is a rare hepatic anatomical variant in which the left hepatic lobe extends into the left upper quadrant and surrounds the spleen. This extension of the left hepatic lobe consists of normal hepatic parenchyma with no functional liver impairment. In trauma cases, however, the extended left hepatic lobe is vulnerable to injury and confused for a splenic injury due to similar echogenicities and densities on ultrasound and CT. It is also misdiagnosed as a splenic subcapsular hematoma, perisplenic hemorrhage, or mass. Usually, the beaver tail liver is encountered incidentally in patients. We present a 67-year-old male with a history of chronic obstructive lung disease, coronary artery disease, myocardial infarction, and aortic valve replacement. The patient was admitted for further evaluation and placed under the Baker Act for attempting to overdose on oxycodone to commit suicide. Initial imaging identified an ill-defined lesion on CT angiography, which raised concerns for potential malignancy of the liver. Ultimately, an MRI of the abdomen ruled out a malignant lesion due to a lack of abnormal contrast enhancement over the circumscribed region. Consequently, further imaging of the liver led to the incidental discovery of the beaver tail liver in this patient. Due to the rarity of this variant, available literature regarding beaver tail liver is limited to several case reports describing it as an incidental finding. This case highlights the rare nature and unique challenges the beaver tail liver presents for emergency medicine physicians, surgeons, and radiologists interpreting imaging studies without knowledge of its existence. It is important to emphasize how the unexpected presence of the left hepatic lobe in the upper left quadrant of the abdomen can lead to misinterpretations in FAST (focused assessment with sonography in trauma) exams and CT scans. Using non-invasive tools, such as color Doppler, is one way to reduce the incorrect diagnosis of hepatic anatomic variants.

Periventricular Leukomalacia Following Bowel Resection for Necrotizing Enterocolitis in a Premature Neonate.

Necrotizing enterocolitis (NEC) and periventricular leukomalacia (PVL) are relatively common conditions in premature infants with low birth weight (VLBW). However, in the current literature, there are limited case reports of patients with concomitant NEC and PVL. We report a case of a premature female born at a gestational age of 25 weeks and five days who developed cystic intracranial lesions after emergent bowel resection due to NEC. Transcranial ultrasound and magnetic resonance imaging confirmed the presence of cystic PVL in the right middle cerebral artery distribution. Several observational studies note the association between spontaneous intestinal perforation, surgical NEC, and the presence of cystic PVL. When infants are unresponsive to medical management for NEC, exploratory laparotomy with resection of the necrotic or perforated intestine is indicated. However, infants treated surgically have poorer neurodevelopmental outcomes than those with medical therapy. Pathogenesis of neurodevelopmental impairment in preterm infants undergoing surgery involves dysfunctional cerebrovascular autoregulation (CAR), which is associated with harmful changes in cerebral perfusion that lead to neuronal injury. Ill preterm infants, such as those with NEC, cannot regulate cerebral perfusion appropriately, and impaired CAR may be present in more than half the preterm infants during laparotomy. Impaired CAR leads to poor cerebral perfusion that potentiates neuronal injury, such as PVL. This case also brings awareness to the need for adherence to screening practices for white matter injury in critical NICU patients through cost-effective transcranial ultrasound.

A comprehensive review of the multifaceted role of the microbiota in human pancreatic carcinoma.

Pancreatic carcinoma is associated with one of the worst clinical outcomes throughout the globe because of its aggressive, metastatic, and drug-resistant nature. During the past decade, several studies have shown that oral, gut, and tumor microbiota play a critical role in the modulation of metabolism and immune responses. Growing pieces of evidence have proved beyond a doubt that the microbiota has a unique ability to influence the tumor microenvironment as well as the metabolism of chemotherapeutic agents or drugs. Given this, microbiota, known as the ecological community of microorganisms, stands to be an avenue of quality research. In this review, we provide detailed and critical information on the role of oral, gut, and pancreatic microbiota disruptions in the development of pancreatic carcinoma. Moreover, we comprehensively discuss the different types of microbiota, their potential role, and mechanism associated with pancreatic carcinoma. The microbiome provides the unique opportunity to enhance the effectiveness of chemotherapeutic agents and immunotherapies for pancreatic cancer by maintaining the right type of microbiota and holds a promising future to enhance the clinical outcomes of patients with pancreatic carcinoma.

Development of polyclonal antibodies using bacterially expressed recombinant coat protein for the detection of Onion yellow dwarf virus (OYDV) and identification of virus free onion genotypes.

Onion yellow dwarf virus (OYDV) belonging to the genus Potyvirus, family Potyviridae, is one of the widely distributed viruses of Allium species worldwide. It causes dwarfing, yellow striping, crinkling and flaccidity of the leaves of onion and garlic. To see the occurrence and incidence of OYDV on Allium crop, an attempt was made to develop antibody based diagnostic assay which would be useful for routine indexing and screening of the germplasm. The total RNA was isolated from the symptomatic leaves of onion and the gene encoding coat protein (CP) was cloned. The nucleotide sequencing analysis of the cloned RT-PCR product revealed ~ 774 bp amplicon (OYDV CP) and it was further cloned in pET-28a ( +) expression vector which yielded ~ 30 kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was primarily checked on SDS-PAGE and further confirmed by Western blot. The His6BP-OYDV-CP was obtained in soluble state after purification and was used to immunize New Zealand white rabbit for the production of polyclonal antibody (PAb). The produced PAb against the purified fusion protein successfully detected OYDV from onion and garlic samples at 1:2000 dilutions in indirect-enzyme linked immunosorbent assay (DAC-ELISA). Thus, this study presents first report that Histidine tag (His6BP) fusion OYDV-CP based antibody production and its successful application in identification of virus free onion and garlic genotypes.

An Intimate Alliance of DNA-Damage Response Network with Cell-Cycle Checkpoints Amid Events of Uncontrolled Cellular Proliferation: A Mini- Review.

There is close interdependence between cell survival, cell senescence, events of the cell cycle, apoptosis, malignancy development, and tumor responses to cancer treatment. Intensive studies and elaborate researches have been conducted on the functional aspects of oncogenes, tumor suppressor genes, apoptotic genes, and members guiding cell cycle regulation. These disquisitions have put forward the existence of a highly organized response pathway termed as a DNA-damage response network. The pathways detecting DNA damage and signaling are intensively linked to the events of cell-cycle arrest, cell proliferation, apoptosis, and cell senescence. DNA damage responses are complex systems that incorporate specific "sensor" and "transducer" proteins, for assessment of damage and signal transmission, respectively. These signals are thereafter relayed upon various "effector" proteins involved in different cellular pathways. It may include those governing cell-cycle checkpoints, participating in DNA repair, cell senescence, and apoptosis. This review discusses the role of the tumour suppressor gene, oncogenes, cell cycle checkpoint regulators during DNA damage response and regulation.

Emerging neo adjuvants for harnessing therapeutic potential of M1 tumor associated macrophages (TAM) against solid tumors: Enusage of plasticity.

Macrophages are a major component of the tumor microenvironment (TME) of most tumors. They are characterized by a high degree of functional plasticity which enable these cells to both promote and eliminate established tumors. Under the influence of immunosuppressive TME, tumor infiltrating iNOS+ and CD11b+ M-1 effector macrophages get polarized towards tumor associated macrophages (TAM) which are tropic to variety of tumors. Increased infiltration and density of TAM is associated with tumor progression and poor prognosis in the plethora of tumors due to their angiogenetic and tissue re-modelling nature. Importantly, TAMs are also responsible for developing endothelium anergy, a major physical barrier for majority of cancer directed immune/chemotherapies. Therefore, functional retuning/re-educating TAM to M-1 phenotypic macrophages is paramount for effective immunotherapy against established tumors. In this review, we discuss and provide comprehensive update on TAM-targeted approaches for enhancing immunity against various solid tumors.

A prospective, randomized comparison of ultrasonographic visualization of proximal intercostal block vs paravertebral block.

BACKGROUND: Thoracic paravertebral blockade is an accepted anesthetic and analgesic technique for breast surgery. However, real-time ultrasound visualization of landmarks in the paravertebral space remains challenging. We aimed to compare ultrasound-image quality, performance times, and clinical outcomes between the traditional parasagittal ultrasound-guided paravertebral block and a modified approach, the ultrasound-guided proximal intercostal block. METHODS: Women with breast cancer undergoing mastectomy (n = 20) were randomized to receive either paravertebral (n = 26) or proximal intercostal blocks (n = 32) under ultrasound-guidance with 2.5 mg/kg ropivacaine prior to surgery. Block ultrasound images before and after needle placement, and anesthetic injection videoclips were saved, and these images and vidoes independently rated by separate novice and expert reviewers for quality of visualization of bony elements, pleura, relevant ligament/membrane, needle, and injectate spread. Block performance times, postoperative pain scores, and opioid consumption were also recorded. RESULTS: Composite visualization scores were superior for proximal intercostal compared to paravertebral nerve block, as rated by both expert (p = 0.008) and novice (p = 0.01) reviewers. Notably, both expert and novice rated pleural visualization superior for proximal intercostal nerve block, and expert additionally rated bony landmark and injectate spread visualization as superior for proximal intercostal block. Block performance times, needle depth, opioid consumption and postoperative pain scores were similar between groups. CONCLUSIONS: Proximal intercostal block yielded superior visualization of key anatomical landmarks, possibly offering technical advantages over traditional paravertebral nerve block. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02911168. Registred on the 22nd of September 2016.

Indibulin dampens microtubule dynamics and produces synergistic antiproliferative effect with vinblastine in MCF-7 cells: Implications in cancer chemotherapy.

Indibulin, a synthetic inhibitor of tubulin assembly, has shown promising anticancer activity with a minimal neurotoxicity in preclinical animal studies and in Phase I clinical trials for cancer chemotherapy. Using time-lapse confocal microscopy, we show that indibulin dampens the dynamic instability of individual microtubules in live breast cancer cells. Indibulin treatment also perturbed the localization of end-binding proteins at the growing microtubule ends in MCF-7 cells. Indibulin reduced inter-kinetochoric tension, produced aberrant spindles, activated mitotic checkpoint proteins Mad2 and BubR1, and induced mitotic arrest in MCF-7 cells. Indibulin-treated MCF-7 cells underwent apoptosis-mediated cell death. Further, the combination of indibulin with an anticancer drug vinblastine was found to exert synergistic cytotoxic effects on MCF-7 cells. Interestingly, indibulin displayed a stronger effect on the undifferentiated neuroblastoma (SH-SY5Y) cells than the differentiated neuronal cells. Unlike indibulin, vinblastine and colchicine produced similar depolymerizing effects on microtubules in both differentiated and undifferentiated SH-SY5Y cells. The data indicated a possibility that indibulin may reduce chemotherapy-induced peripheral neuropathy in cancer patients.

Protein Nanoparticles: Promising Platforms for Drug Delivery Applications.

The development of drug delivery systems using nanoparticles as carriers for small and large therapeutic molecules remains a rapidly growing area of research. The advantages of using proteins to prepare nanoparticles for drug delivery applications include their abundance in natural sources, biocompatibility, biodegradability, easy synthesis process, and cost-effectiveness. In contrast to several particulate systems like nanoparticles from metallic and inorganic/synthetic sources, the protein nanoparticles do not have limitations such as potential toxicity, large size, accumulation, or rapid clearance from the body. In addition, protein-based nanoparticles offer the opportunity for surface modification by conjugation of other protein and carbohydrate ligands. This enables targeted delivery to the desired tissue and organ, which further reduces systemic toxicity. The use of protein nanoparticles for such applications could therefore prove to be a better alternative to maneuver and improve the pharmacokinetic and pharmacodynamic properties of the various types of drug molecules. In this review, while focusing on the properties of a few proteins such as the silk protein fibroin, we attempt to provide an overview of the existing protein-based nanoparticles. We discuss various methods for the synthesis of this class of nanoparticles. The review brings forth some of the factors that are important for the design of this class of nanoparticles and highlights the applications of the nanoparticles obtained from these proteins.

Enhanced stability of microtubules contributes in the development of colchicine resistance in MCF-7 cells.

Understanding the mechanism of resistance to tubulin-targeted anticancer drugs is important for improved chemotherapy. In this work, a colchicine-resistant MCF-7 cell line (MCF-7Col30) was generated by the gradual increment of colchicine treatment and the MCF-7Col30 showed ∼8-fold resistance towards colchicine. MCF-7Col30 cells showed ∼2.5-fold resistance against microtubule depolymerizing agents, vinblastine, and nocodazole. In contrast, it displayed more sensitivity towards paclitaxel, a microtubule-polymerizing agent. MCF-7 and MCF-7Col30 cells showed similar sensitivity towards cisplatin. Further, the level of P-glycoprotein did not increase in MCF-7Col30 cells. MCF-7Col30 cells resisted the microtubule depolymerizing effects of colchicine. The time-lapse imaging of individual microtubules in live cells showed that the dynamics of microtubules in MCF-7Col30 cells was suppressed as compared to the parent MCF-7 cells. The levels of tubulin acetylation and glutamylation increased in MCF-7Col30 cells than the parent MCF-7 cells suggesting that microtubules are stabilized in MCF-7Col30 cells. Interestingly, the level of ßIII tubulin was increased by 2.3 folds whereas that of ßII and ßIV tubulin was decreased by 55 and 150%, respectively in MCF-7Col30 cells. The results suggested that the changes in the level of ß-tubulin isoforms and the post-translational modifications of microtubules altered the stability and dynamics of microtubules and contributed to the development of colchicine-resistance in MCF-7 cells.

Identification of NURR1 (Exon 4) and FOXA1 (Exon 3) Haplotypes Associated with mRNA Expression Levels in Peripheral Blood Lymphocytes of Parkinson's Patients in Small Indian Population.

Here, we study the expression of NURR1 and FOXA1 mRNA in peripheral blood lymphocytes and its haplotypes in coding region in a small Chennai population of India. Thirty cases of Parkinson's patients (PD) with anti-PD medications (20 males aged 65.85 ± 1.19 and 10 females aged 65.7 ± 1.202) and 30 age matched healthy people (20 males aged 68.45 ± 1.282 and 10 females aged 65.8 ± 1.133) were included. The expression of NURR1 and FOXA1 in PBL was detected by Q-PCR and haplotypes were identified by PCR-SSCP. In the 30 PD cases examined, NURR1 and FOXA1 expression was significantly reduced in both male and female PD patients. However, NURR1 (57.631% reduced in males; 28.93% in females) and FOXA1 (64.42% in males; 55.76% in females) mRNA expression did differ greatly between male and female PD patients. Polymorphisms were identified at exon 4 of the NURR1 and at exon 3 of the FOXA1, respectively, in both male and female patients. A near significant difference in SSCP patterns between genders of control and PD population was analyzed suggesting that further investigations of more patients, more molecular markers, and coding regions should be performed. Such studies could potentially reveal peripheral molecular marker of early PD and different significance to the respective genders.

Silk protein-based hydrogels: Promising advanced materials for biomedical applications.

Hydrogels are a class of advanced material forms that closely mimic properties of the soft biological tissues. Several polymers have been explored for preparing hydrogels with structural and functional features resembling that of the extracellular matrix. Favourable material properties, biocompatibility and easy processing of silk protein fibers into several forms make it a suitable material for biomedical applications. Hydrogels made from silk proteins have shown a potential in overcoming limitations of hydrogels prepared from conventional polymers. A great deal of effort has been made to control the properties and to integrate novel topographical and functional characteristics in the hydrogel composed from silk proteins. This review provides overview of the advances in silk protein-based hydrogels with a primary emphasis on hydrogels of fibroin. It describes the approaches used to fabricate fibroin hydrogels. Attempts to improve the existing properties or to incorporate new features in the hydrogels by making composites and by improving fibroin properties by genetic engineering approaches are also described. Applications of the fibroin hydrogels in the realms of tissue engineering and controlled release are reviewed and their future potentials are discussed. STATEMENT OF SIGNIFICANCE: This review describes the potentiality of silk fibroin hydrogel. Silk Fibroin has been widely recognized as an interesting biomaterial. Due to its properties including high mechanical strength and excellent biocompatibility, it has gained wide attention. Several groups are exploring silk-based materials including films, hydrogels, nanofibers and nanoparticles for different biomedical applications. Although there is a good amount of literature available on general properties and applications of silk based biomaterials, there is an inadequacy of extensive review articles that specifically focus on silk based hydrogels. Silk-based hydrogels have a strong potential to be utilized in biomedical applications. Our work is an effort to highlight the research that has been done in the area of silk-based hydrogels. It aims to provide an overview of the advances that have been made and the future course available. It will provide an overview of the silk-based hydrogels as well as may direct the readers to the specific areas of application.

The Sojourn from Parenteral to Oral Taxanes using Nanocarrier Systems: A Patent Review.

Natural diterpenoid taxanes and their semisynthetic analogues have already made an indelible mark in the chemotherapeutic world for treating various kinds of malignancies. However, due to the absence of any functional groups which could be ionized by pH alteration or which may participate in salt formation, these taxanes, exhibit low solubility. Parenteral administration of taxanes with solubilising agents such as Cremophor EL and Polysorbate 80 results in undesirable side effects like hypersensitivity reactions, myelosuppression and peripheral neuropathy. Nanoengineered drug delivery systems like nanoemulsions, nanocrystals, dendrimers, micelles, selfnanoemulsifying systems, liposomes, solid-lipid nanoparticles and biodegradable polymeric nanoparticles, in this regard, tend to surmount these ostensible challenges, when administered orally. The bare taxanes encounter several inadequacies, namely poor aqueous solubility, structural instability in physiological fluids, p-glycoprotein recognition, hepatic first-pass effect, gastrointestinal permeability, and Cytochrome P450 enzymatic metabolism, etc. In addition, nanoscaled oral delivery improves drug encapsulation, thus facilitating diffusion through intestinal epithelium, modification of pharmacokinetic and tissue distribution profile of the drug, eventually resulting in flexibility of dosing schedules, prevention of discomfort of the injection and hospitalization, and improved patient convenience. The current review paper endeavors to provide a bird's eye view on the significant headway made on orally-administered nanosystems of taxanes and their analogues through patent applications published till date since its discovery.

Quantum Dots and their Potential Role in Cancer Theranostics.

The emergence of cancer nanomedicine is the result of fruitful advances in the fields of nanotechnology, bioimaging, formulation development, and molecular biology. Quantum dots (QDs) are the luminescent nanocrystals (NCs) that provide a multifunctional platform for imaging the biosystems following controlled delivery of therapeutic drugs, proteins, peptides, oligonucleotides, and genes. These engineered fluorescent probes with integrated imaging and carrier functionalities have become excellent tools for molecular diagnostics and delivery of therapeutics molecules. Flexible surface chemistry, unique optical properties, high sensitivity, and multiplexing capabilities of QDs certainly make them a most promising tool for personalized medicine. This review focuses on state-of-art advances in synthesizing QDs and highlights the approaches used for functionalization of QDs with desired ligands for targeted carriage to specific sites. Discussed is the role of QDs in antitumor therapy through drug delivery and gene delivery and the recently emerged photodynamic therapy (PDT). We also endeavor to critically address the major impediments in the clinical development of these multifunctional nanoplatforms, with a special focus on plausible advancements for the near future.

Sesame (Sesamum indicum L.).

Sesame (Sesamum indicum L.) is an important oilseed crop grown in India, China, Korea, Russia, Turkey, Mexico, South America, and several countries of Africa. Sesame seeds are rich in oil, proteins, unsaturated fatty acids, vitamins, minerals, and folic acid. Nearly 70% of the world's sesame is processed into oil and meal, while the remainder is channeled to food and confectionery industries. Production of sesame is limited by several fungal diseases, water logging, salinity, and shattering of seed capsules during harvest. Introgression of useful genes from wild species into cultigens by conventional breeding has not been successful due to postfertilization barriers. The only alternative for the improvement of S. indicum is to transfer genes from other sources through genetic transformation techniques. Here, we describe a simple, fast, and reproducible method for the Agrobacterium-mediated genetic transformation of S. indicum which may be employed for the transfer of desirable traits into this economically important oilseed crop.

Transcription factor NF-κB associates with microtubules and stimulates apoptosis in response to suppression of microtubule dynamics in MCF-7 cells.

NF-κB, a master regulator of several signaling cascades, is known to be actively transported in the nucleus in response to various stimuli. Here, we found that NF-κB is associated with polymeric tubulin and co-localized with microtubules in MCF-7 cells. Using TN16, a known microtubule targeting agent, we found that microtubule dynamics plays a critical role in NF-κB-microtubule interaction. Treatment of cells with low concentrations of TN16 (25 and 50 nM) that suppressed microtubule dynamics without visibly affecting microtubule organization enhanced the association of NF-κB with microtubules and facilitated nuclear translocation of NF-κB. Colchicine and vinblastine also produced similar nuclear translocation of NF-κB. Further, nuclear import of NF-κB activated apoptotic pathway in the cells that were blocked in mitosis by TN16 treatment suggesting that NF-κB acts as a pro-apoptotic protein in response to the suppression of microtubule dynamics. Interestingly, in the presence of high concentrations of TN16 that extensively disrupted the microtubule network, though there was an increase in the apoptotic cell death, the interaction of NF-κB with microtubules and its nuclear import were significantly reduced. Under these conditions, we detected an increase in the level of phosphorylation and nuclear accumulation of ERK, a MAP kinase, suggesting that the induction of apoptosis was caused by ERK signaling. The results indicate that the interaction of NF-κB with microtubules, its nuclear accumulation and subsequent gene transcription are critically dependent on microtubule dynamics. The data suggest a correlation between the functional status of microtubules and different apoptotic mechanisms invoked in response to microtubule inhibitors.

Inhibition of HDAC6 deacetylase activity increases its binding with microtubules and suppresses microtubule dynamic instability in MCF-7 cells.

The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ε-amino group of Lys-40 of α-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions.

Kinetic stabilization of microtubule dynamics by indanocine perturbs EB1 localization, induces defects in cell polarity and inhibits migration of MDA-MB-231 cells.

Cell motility is an essential aspect of metastatic spread of cancer. Microtubule-targeted agents exhibit anti-metastatic properties, the underlying mechanism of which remains understudied. In this study, we have investigated the role of microtubule dynamics in migration of cancer cells using indanocine, a synthetic small molecule inhibitor of tubulin. We found that indanocine, at concentrations that did not visibly affect microtubule organization, suppressed dynamic instability of microtubules and reduced the rate of migration of highly metastatic MDA-MB-231 cells. Indanocine-treated cells were defective in lamellipodium formation and could not develop polarized morphology. The kinetic stabilization of microtubules was associated with a marked increase in their acetylation level and a perturbation in the localization of EB1, a microtubule plus end binding protein. Using standard scratch wound healing assay and immunofluorescence analysis; we found that microtubule acetylation occurred in the direction of migration in vehicle-treated cells, whereas indanocine treatment led to a global acetylation of microtubules. The results together suggested that selective stabilization of microtubules was perturbed in the presence of indanocine that possibly resulted in lack of cell polarization and a concurrent reduction in migration of cells. Moreover, microtubule stabilization by indanocine affected adhesion turnover and impaired the polarized pattern of adhesion sites in cells. Together the results indicated that the regulation of microtubule dynamics is required to coordinate cell polarization as well as adhesion asymmetry and support the hypothesis that the perturbation of microtubule dynamics by tubulin-targeted agents can be exploited to restrict the migration of tumor cells.