Molecular structure of cotinine studied by gas electron diffraction combined with theoretical calculations

The molecular structure of cotinine ((S)-1-methyl-5-(3-pyridinyl)-2-pyrrolidinone), the major metabolite of nicotine, has been determined at about 182 °C by gas electron diffraction combined with MP2 and DFT calculations. The diffraction data are consistent with the existence of the (ax, sc), (ax, ap), (eq, sp) and (eq, ap) conformers, where ax and eq indicate the configuration of the pyrrolidinone ring by means of the position (axial and equatorial) of the pyridine ring, and sc, sp and ap distinguish the isomers arising from the internal rotation around the bond connecting the two rings. The (CH3)NCCC(N) dihedral angles, , of the (ax, sc) and (eq, sp) conformers were determined independently to be 158(12)° and 129(13)°, respectively, where the numbers in parentheses are three times the standard errors, 3σ. According to the MP2 calculations, the corresponding dihedral angles for the (ax, ap) and (eq, ap) conformers were assumed to differ by 180° from their syn counterparts. The ratios x(ax, sc)/x(ax, ap) and x(eq, sp)/x(eq, ap) were taken from the theoretically estimated free energy differences, ΔG, where x is the abundance of the conformer. The resultant abundances of (ax, sc), (ax, ap), (eq, sp) and (eq, ap) conformers are 34(6)%, 21% (d.p.), 28% (d.p.), and 17% (d.p.), respectively, where d.p. represents dependent parameters. The determined structural parameters (rg(Å) and ∠α(°)) of the most abundant conformer, (ax, sc), are as follows: r(N–C)pyrrol = 1.463(5); r(N–Cmethyl) = 1.457(←); r(N–C(=O))= 1.384(12); r(C=O)= 1.219(5); = 1.541(3); r(Cpyrrol–Cpyrid) = 1.521(←); = 1.396(2); = 1.343(←); ∠(CNC)pyrrol= 113.9(11); ∠CCCpyrrol(–Cpyrid) = 103.6(←); ∠NCO = 124.1(13); ∠NCpyrrolCpyrid = 113.1(12); ∠CpyrrolCpyrrolCpyrid = 113.3(←); ∠(CNC)pyrid = 117.1(2); = 124.4(←); ∠CmethylNC(=O) = ∠CmethylNC(–Cpyrid) = 122.8(d.p.); ∠ΝC(=O)C = 107.1(d.p.); ∠NCpyrrol(–Cpyrid)Cpyrrol = 103.0(d.p.) and ∠CCC(=O) =105.2(d.p.), where ← in the parentheses means that the parameter is bound to the preceding one and denote average values. The puckering angle, α, of the pyrrolidinone ring is 26(3)°.The N…N distances of the (ax, sc) and (eq, sp) conformers, which are 4.844(5) and 4.740(5) Å, respectively, are close to that of the most stable conformer of nicotine, 4.885(6) Å and the corresponding one of arecoline, 4.832(13) Å. It is concluded that the weak nicotinic activity of cotinine cannot be ascribed to such a small difference in the N…N distances

The analysis of Radiation-induced, tumor-infiltrating neutrophils, which has the antitumor characteristics

It is now becoming clear that interactions between tumor cells and host tissue stoma play a key role in tumor progression. Understanding the composition of the stromal cells in the tumor microenvironment immediately after tumor irradiation might be an important first step in immunomodulation by radiation therapy. To explore this, we harvested tumor mass, draining lymph nodes (DLNs), spleen and peripheral blood mononuclear cells (PBMCs) at different times after a single 15 Gy dose of focused irradiation of RM-9 mouse prostate tumor grafts growing in the hind leg of syngeneic C57BL/6 mice. Subpopulations of lymphocytes and granulocytes (CD4+, CD8+, CD4+CD25+, CD11c+, CD11b+Gr-1+mid and CD11b+Gr-1+ highLy-6G+ cells) were analyzed in the harvested tissue by flow cytometry. An infiltration of CD11b+Gr-1+ highLy-6G+ neutrophils reached a peak within the tumor microenvironment at 24 h after tumor irradiation while no significant changes in the other subpopulations were noted. Increased neutrophil infiltration after irradiation was also observed when the mammary gland cancer cell line 4T1 was implanted in BALB/c mice. To investigate the effect of neutrophils on tumor growth, we compared the tumor size in mice treated with the neutrophil-depleting anti-Ly-6G monoclonal antibody (Mab) to that of mice treated with an isotype –matched control antibody. Interestingly, in RM-9, and 4T1 tumor models, the therapeutic effect of irradiation was significantly attenuated in the mice with depleted neutrophils. To evaluate whether the early infiltration of neutrophils had significant tumor-specific immune response, we used EG7 (OVA gene-transfected)-tumor-bearing mice for detecting OVA-specific CTL. Then, these tumors (with and without neutrophil depletion by anti-Ly-6G MAb) were irradiated focally with 15 Gy. 7 days after irradiation, we observed the frequency of OVA-tetramer+CD8+ cells (OVA-specific CD8+) in tumor-bearing mice. A decrease in OVA-tetramer+CD8+ cells was observed in the DLN of the neutrophil-depleted mice. This result is consistent with the tumor growth curve. Hence neutrophil infiltration might be a very early but important step in initiating a robust adaptive tumor specific response in the DLN. Now we are investing how this neutrophil works for inducing tumor-specific CTL and for showing antitumor effect.The 7th GI-CoRE Medical Science and Engineering Symposiu

放射線治療後に腫瘍内に浸潤する好中球は抗腫瘍作用を示す

Radiation therapy (RT) is one of the primary treatment modalities for many cancers. In the present study using the RM-9 (mouse prostate tumor cell line)-bearing C57BL/6 mice or to the 4T1(mouse mammary cell line)-bearing BALB/c mice, we demonstrated that RT induces sterile inflammation with an infiltration of CD11b+Gr-1+ neutrophils reached a peak within the tumor microenvironment at 24-48 h after tumor RT. RT-recruited neutrophils (RT-Ns) exhibit an increased production of reactive oxygen species (ROS) and induce apoptosis of tumor cells. The anti-tumor activity of RT is significantly reduced when RT-Ns are depleted with an anti-Ly-6G antibody (1A8), resulting in decreased apoptosis of tumor cells. In contrast, treatment with G-CSF, known to activate neutrophils, elevates ROS production by RT-Ns. This leads to enhanced tumor cytotoxicity followed by an increased tumor-specific CD8+ T cell response. These results suggest that RT given in conjunction with G-CSF may be an effective strategy for improving the anti-tumor activity of RT.第78回日本癌学会学術総

Selective targeting of cancerous mitochondria and suppression of tumor growth using redox-active treatment adjuvant

Redox-active substances and their combinations, such as of quinone/ascorbate, and in particular menadione/ascorbate (M/A; also named Apatone attract attention with their unusual ability to kill cancer cells without affecting the viability of normal cells, as well as with the synergistic anticancer effect of both molecules. So far, the primary mechanism of M/A-mediated anticancer effects has not been linked to mitochondria. The aim of our study was to clarify whether this “combination drug” affects mitochondrial functionality specifically in cancer cells. Studies were conducted on cancer cells (Jurkat, Colon26, MCF7) and normal cell (normal lymphocytes, FHC, MCF10A), treated with different concentrations of menadione, ascorbate and/or their combination (2/200, 3/300, 5/500, 10/1000, and 20/2000 M/M of M/A). M/A exhibited highly specific and synergistic suppression on cancer cell growth, but without adversely affecting the viability of normal cells at pharmacologically attainable concentrations. In M/A-treated cancer cells, the cytostatic/cytotoxic effect is accompanied by: (i) extremely high production of mitochondrial superoxide (up to 15 fold over the control level); (ii) a significant decrease of mitochondrial membrane potential; (iii) a decrease of the steady-state levels of ATP, succinate, NADH, and NAD+; and (iv) a decreased expression of programed cell death ligand 1 (PD-L1) – one of the major immune checkpoints. These effects were dose-dependent. Inhibition of NQO1 by dicoumarol increased mitochondrial superoxide and sensitized cancer cells to M/A. In normal cells, M/A induced relatively low and dose-independent increase of mitochondrial superoxide and mild oxidative stress, which seems to be well tolerated. These data suggest that all anticancer effects of M/A result from a specific mechanism, tightly connected to the mitochondria of cancer cells. At low/tolerable doses of M/A (1/100-3/300 M/M) attainable in cancer by oral and parenteral administration, M/A sensitized cancer cells to conventional anticancer drugs, exhibiting synergistic or additive cytotoxicity accompanied by impressive induction of apoptosis. Combinations of M/A with 13 anticancer drugs were investigated (ABT-737, Barasertib, Bleomycin, BEZ-235, Bortezomib, Cisplatin, Everolimus, Lomustin, Lonafarnib, MG-132, MLN-2238, Palbociclib, PI-103).Low/tolerable doses of M/A did not induce irreversible cytotoxicity in cancer cells, but did cause irreversible metabolic changes, including: (i) a decrease of succinate and NADH; (ii) depolarization of the mitochondrial membrane; and (iii) overproduction of superoxide in the mitochondria of cancer cells only. In addition, M/A suppressed tumor growth in vivo after oral administration in mice with melanoma and the drug down-regulated PD-L1 in melanoma cells. Experimental data suggest a great potential for beneficial anticancer effects of M/A through increasing the sensitivity of cancer cells to conventional anticancer therapy, as well as to the immune system, while sparing normal cells. We hypothesize that M/A-mediated anticancer effects are triggered by redox-cycling of both substances, specifically within dysfunctional mitochondria. M/A may also have a beneficial effect on the immune system, making cancer cells "visible" and more vulnerable to the native immune response