Interplay of phosphorus doses, cyanobacterial inoculation, and elevated carbon dioxide on yield and phosphorus dynamics in cowpea.

Phosphorus (P) demand is likely to increase especially in legumes to harness greater benefits of nitrogen fixation under elevated CO2 condition. In the following study, seed yield and seed P uptake in cowpea increased by 26.8% and 20.9%, respectively, under elevated CO2 level. With an increase in phosphorus dose up to 12 mg kg-1, seed yield enhanced from 2.6 to 5.4 g plant-1. P application and cyanobacterial inoculation increased the microbial activity of soil, leading to increased availability of P. Under elevated CO2 condition, microbial activity, measured as dehydrogenase, acid phosphatase, and alkaline phosphatase activities showed stimulation. Soil available P also increased under elevated CO2 condition and was stimulated by both P application and cyanobacterial inoculation. Higher P uptake in elevated CO2 condition led to lower values of inorganic P in soil. Stepwise regression analysis showed that aboveground P uptake, soil available P, and alkaline phosphatase activity of soil influenced the yield while available P, and organic and inorganic P influenced the aboveground P uptake of the crop. This study revealed that under elevated CO2 condition, P application and cyanobacterial inoculation facilitated P uptake and yield, mediated through enhanced availability of nutrients, in cowpea crop.

Gold (I) N-heterocyclic carbene complex inhibits mouse melanoma growth by p53 upregulation.

BACKGROUND: Cancer treatment using gold (I) complexes is becoming popular. In this study, a gold (I) N-heterocyclic complex designated as complex 3 was synthesized, its cytotoxicity was examined, and its anti-melanoma activity was evaluated in vitro and in vivo. METHODS: Viability of cancer cells was determined by MTT assay upon treatment with various concentrations of a gold (I) N-heterocyclic carbene complex (complex 3) in a dose and time dependent manner. Mouse melanoma cells B16F10 were selected for further apoptotic studies, including flowcytometric analysis of annexin binding, cell cycle arrest, intracellular ROS generation and loss in the mitochondrial membrane potential. ELISA based assays were done for caspase activities and western blots for determining the expression of various survival and apoptotic proteins. Immunocytology was performed to visualize the translocation of p53 to the nucleus. B16F10 cells were inoculated into mice and post tumor formation, complex 3 was administered. Immunohistology was performed to determine the expressions of p53, p21, NF-κB (p65 and p50), MMP-9 and VEGF. Student's t test was used for determining statistical significance. The survival rate data were analyzed by Kaplan-Meier plots. RESULTS: Complex 3 markedly inhibited the growth of HCT 116, HepG2, and A549, and induced apoptosis in B16F10 cells with nuclear condensation, DNA fragmentation, externalization of phosphatidylserine, activation of caspase 3 and caspase 9, PARP cleavage, downregulation of Bcl-2, upregulation of Bax, cytosolic cytochrome c elevation, ROS generation, and mitochondrial membrane potential loss indicating the involvement of an intrinsic mitochondrial death pathway. Further, upregulation of p53, p-p53 (ser 15) and p21 indicated the role of p53 in complex 3 mediated apoptosis. The complex reduced tumor size, and caused upregulation of p53 and p21 along with downregulation of NF-κB (p65 and p50), VEGF and MMP-9. These results suggest that it induced anti-melanoma effect in vitro and in vivo by modulating p53 and other apoptotic factors. CONCLUSIONS: The gold (I) N-heterocyclic carbene complex (C22H26N6AuO2PF6) designated as complex 3 induced ROS and p53 dependent apoptosis in B16F10 cells involving the mitochondrial death pathway along with suppression of melanoma tumor growth by regulating the levels of pro and anti apoptotic factors (p53, p21, NF-κB, VEGF and MMP-9).

Novel selective inhibitors of macropinocytosis-dependent growth in pancreatic ductal carcinoma.

Macropinocytosis is a cellular process that enables cells to engulf extracellular material, such as nutrients, growth factors, and even whole cells. It is involved in several physiological functions as well as pathological conditions. In cancer cells, macropinocytosis plays a crucial role in promoting tumor growth and survival under nutrient-limited conditions. In particular KRAS mutations have been identified as main drivers of macropinocytosis in pancreatic, breast, and non-small cell lung cancers. We performed a high-content screening to identify inhibitors of macropinocytosis in pancreatic ductal adenocarcinoma (PDAC)-derived cells, aiming to prevent nutrient scavenging of PDAC tumors. The screening campaign was conducted in a well-known pancreatic KRAS-mutated cell line (MIAPaCa-2) cultured under nutrient deprivation and using FITC-dextran to precisely quantify macropinocytosis. We assembled a collection of 3584 small molecules, including drugs approved by the Food and Drug Administration (FDA), drug-like molecules against molecular targets, kinase-targeted compounds, and molecules designed to hamper protein-protein interactions. We identified 28 molecules that inhibited macropinocytosis, with potency ranging from 0.4 to 29.9 µM (EC50). A few of them interfered with other endocytic pathways, while 11 compounds did not and were therefore considered specific "bona fide" macropinocytosis inhibitors and further characterized. Four compounds (Ivermectin, Tyrphostin A9, LY2090314, and Pyrvinium Pamoate) selectively hampered nutrient scavenging in KRAS-mutated cancer cells. Their ability to impair albumin-dependent proliferation was replicated both in different 2D cell culture systems and 3D organotypic models. These findings provide a new set of compounds specifically targeting macropinocytosis, which could have therapeutic applications in cancer and infectious diseases.

Role of Red-Ox Cycle in Structural Oscillations and Solvation Dynamics in the Mitochondria of a Live Cell.

Structural oscillations and solvation dynamics in the mitochondria of a live cell are studied by time-resolved microscopy using a covalent fluorescence probe. We compared the dynamics in a human breast cancer cell (MCF-7) with that in a normal breast cell MCF-10A. The probe, CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin), binds with the free thiol groups. In MCF-10A cell, CPM binds with the discrete mitochondria. In MCF-7, CPM labels the clustered mitochondria in the peri-nuclear region. Location of the CPM in the mitochondria is confirmed by colocalization with a mitochondria-tracker dye. The red-ox cycle in the mitochondria causes periodic fluctuation in the microenvironment in the discrete mitochondria. This is manifested in fluctuations in fluorescence intensity of CPM bound to mitochondria. The magnitude of oscillation is much less for CPM bound to the clustered mitochondria (in which the red-ox cycle is inefficient) in the cancer cell (MCF-7). In both of the cells (MCF-10A and MCF-7) CPM bound to thiol-containing proteins in mitochondria exhibits ultraslow response with average solvation time (⟨τs⟩) of 850 and 1400 ps in MCF-10A and MCF-7, respectively.

Cytotoxic activity and apoptosis-inducing potential of di-spiropyrrolidino and di-spiropyrrolizidino oxindole andrographolide derivatives.

Anticancer role of andrographolide is well documented. To find novel potent derivatives with improved cytotoxicity than andrographolide on cancer cells, two series of di-spiropyrrolidino- and di-spiropyrrolizidino oxindole andrographolide derivatives prepared by cyclo-addition of azomethine ylide along with sarcosine or proline (viz. sarcosine and proline series respectively) and substitution of different functional groups (-CH3, -OCH3 and halogens) were examined for their cytotoxic effect on a panel of six human cancer cell lines (colorectal carcinoma HCT116 cells, pancreatic carcinoma MiaPaCa-2 cells, hepatocarcinoma HepG2 cells, cervical carcinoma HeLa cells, lung carcinoma A549 and melanoma A375 cells). Except halogen substituted derivatives of proline series (viz. CY2, CY14 and CY15 for Br, Cl and I substitution respectively), none of the other derivatives showed improved cytotoxicity than andrographolide in the cancer cell lines examined. Order of cytotoxicity of the potent compounds is CY2>CY14>CY15>andrographolide. Higher toxicity was observed in HCT116, MiaPaCa-2 and HepG2 cells. CY2, induced death of HCT116 (GI50 10.5), MiaPaCa-2 (GI50 11.2) and HepG2 (GI50 16.6) cells were associated with cell rounding, nuclear fragmentation and increased percentage of apoptotic cells, cell cycle arrest at G1 phase, ROS generation, and involvement of mitochondrial pathway. Upregulation of Bax, Bad, p53, caspases-3,-9 and cleaved PARP; downregulation of Bcl-2, cytosolic NF-κB p65, PI3K and p-Akt; translocation of P53/P21, NF-κB p65 were seen in CY2 treated HCT116 cells. Thus, three halogenated di-spiropyrrolizidino oxindole derivatives of andrographolide are found to be more cytotoxic than andrographolide in some cancer cells. The most potent derivative, CY2 induced death of the cancer cells involves ROS dependent mitochondrial pathway like andrographolide.