Page 244 - 2014 Printable Abstract Book
P. 244
(PS4-16) Triptolide mitigates radiation-induced pneumonitis and inhibits TNF-α and KC. Shanmin Yang,
MD; Mei Zhang, MD; Zhenhuan Zhang, MD, PhD; Liangjie Yin, MS; Steven B. Zhang, DVM, PhD; Lurong
Zhang, MD, PhD; and Paul Okunieff, MD, University of Florida Shands Cancer Center, Gainesville, FL
Introduction: We previously demonstrated that triptolide (TPL) prevents radiation-induced
pneumonitis and fibrosis. We hypothesize that TPL inhibits irradiated macrophages from secreting tumor
necrosis factor-alpha (TNF-α) that augments KC secretions from the lung epithelia, thereby attenuating
pulmonary inflammatory responses. Methods: MLE-15 (lung epithelial) and RAW 264.7 (macrophage)
cells were cultured singly or together in 24-well plates. Pulmonary irradiated C57BL/6 mice were treated
with TPL (0.25 mg/kg, twice a week) by tail vein injection. Pathology and cytokine analysis were performed
on lung tissues. White blood cell counts were performed on lung lavage fluid.
Results: MLE-15 cells produced KC but not TNF-α, and KC was slightly upregulated after irradiation.
Neither irradiated nor nonirradiated RAW 264.7 cells produced KC; however, TNF-α secretion from
irradiated Raw 264.7 cells was increased. In contrast, KC production in MLE-15 cells was augmented by
conditional medium from irradiated Raw 264.7 cells, co-cultured irradiated RAW 264.7 cells, and murine
rTNF-α. Anti-TNF Ab attenuated the effect of conditioned medium. Low concentrations of TPL attenuated
KC production in MLE-15 cells cultured with conditional medium, co-cultured irradiated Raw 264.7 cells,
and after addition of rTNF-α. TPL inhibited the secretion of KC and TNF-α in homogenates of irradiated
lungs. TPL also reduced the infiltration of white blood cells in lavage fluid in the lungs of irradiated C57BL/6
mice. Pathological sections of lungs from the irradiated mice revealed less infiltration of inflammatory
cells than the vehicle alone group. Conclusions: TNF-α augments KC production in MLE-15 cells co-
cultured with irradiated Raw 264.7 cells. TNF-α from pulmonary macrophages appears to induce KC from
the epithelium, which in turn induces inflammatory infiltrates and pneumonitis. TPL inhibits TNF-α and KC
secretions in co-culture in vitro; this is consistent with our in vivo pulmonary irradiated mouse model.
(PS4-17) Xanthone derivative R71 inhibits cancer cell growth and enhances radiosensitivity. Chaomei
Liu; Mei Zhang, MD; Zhenhuan Zhang, MD, PhD; Steven B. Zhang, DVM, PhD; Shanmin Yang, MD; Steven
G. Swarts, PhD; Sadasivan Vidyasagar, MD, PhD; Lurong Zhang, MD, PhD; and Paul Okunieff, MD,
University of Florida Shands Cancer Center, Gainesville, FL
Objective: Xanthone derivatives are produced by a number of plants and have been reported to
have diverse biological profiles, including anticancer, anti-inflammatory, and antioxidant effects. We
synthesized many derivatives to optimize these anticancer and radioprotective effects. We report here
on 3,6-bis (2-oxiranylmethoxy)- 9H-xanthen-9-one, designated R71. Methods: Compound R71 was
1
synthesized beginning with 2, 4-dimethoxybenzoic acid. The structure of R71 was confirmed by H-NMR
13
and C-NMR. The antiproliferative effect of R71 in several cancer cell lines, including A549 (lung
cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer), was
measured by MTT assay. Different concentrations of R71 were added to the cell culture system for 72
hours and evaluated using the MTT assay. Apoptosis and mitochondrial stability were assayed on A549
cells using flow cytometry and with JC-1 and ROS probes. Assays were performed with and without
treatment with various doses of radiation and cisplatin. Data were analyzed with CompuSyn software.
Results: The IC50 of R71 was 19.87, 24.68, 10.3, and 13.09 μM, respectively, in the 4 cancer cell lines
tested. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R71 induced apoptosis
in A549 cells. JC-1 staining of A549 cells showed unstable mitochondrial membrane potential after R71
242 | P a g e
MD; Mei Zhang, MD; Zhenhuan Zhang, MD, PhD; Liangjie Yin, MS; Steven B. Zhang, DVM, PhD; Lurong
Zhang, MD, PhD; and Paul Okunieff, MD, University of Florida Shands Cancer Center, Gainesville, FL
Introduction: We previously demonstrated that triptolide (TPL) prevents radiation-induced
pneumonitis and fibrosis. We hypothesize that TPL inhibits irradiated macrophages from secreting tumor
necrosis factor-alpha (TNF-α) that augments KC secretions from the lung epithelia, thereby attenuating
pulmonary inflammatory responses. Methods: MLE-15 (lung epithelial) and RAW 264.7 (macrophage)
cells were cultured singly or together in 24-well plates. Pulmonary irradiated C57BL/6 mice were treated
with TPL (0.25 mg/kg, twice a week) by tail vein injection. Pathology and cytokine analysis were performed
on lung tissues. White blood cell counts were performed on lung lavage fluid.
Results: MLE-15 cells produced KC but not TNF-α, and KC was slightly upregulated after irradiation.
Neither irradiated nor nonirradiated RAW 264.7 cells produced KC; however, TNF-α secretion from
irradiated Raw 264.7 cells was increased. In contrast, KC production in MLE-15 cells was augmented by
conditional medium from irradiated Raw 264.7 cells, co-cultured irradiated RAW 264.7 cells, and murine
rTNF-α. Anti-TNF Ab attenuated the effect of conditioned medium. Low concentrations of TPL attenuated
KC production in MLE-15 cells cultured with conditional medium, co-cultured irradiated Raw 264.7 cells,
and after addition of rTNF-α. TPL inhibited the secretion of KC and TNF-α in homogenates of irradiated
lungs. TPL also reduced the infiltration of white blood cells in lavage fluid in the lungs of irradiated C57BL/6
mice. Pathological sections of lungs from the irradiated mice revealed less infiltration of inflammatory
cells than the vehicle alone group. Conclusions: TNF-α augments KC production in MLE-15 cells co-
cultured with irradiated Raw 264.7 cells. TNF-α from pulmonary macrophages appears to induce KC from
the epithelium, which in turn induces inflammatory infiltrates and pneumonitis. TPL inhibits TNF-α and KC
secretions in co-culture in vitro; this is consistent with our in vivo pulmonary irradiated mouse model.
(PS4-17) Xanthone derivative R71 inhibits cancer cell growth and enhances radiosensitivity. Chaomei
Liu; Mei Zhang, MD; Zhenhuan Zhang, MD, PhD; Steven B. Zhang, DVM, PhD; Shanmin Yang, MD; Steven
G. Swarts, PhD; Sadasivan Vidyasagar, MD, PhD; Lurong Zhang, MD, PhD; and Paul Okunieff, MD,
University of Florida Shands Cancer Center, Gainesville, FL
Objective: Xanthone derivatives are produced by a number of plants and have been reported to
have diverse biological profiles, including anticancer, anti-inflammatory, and antioxidant effects. We
synthesized many derivatives to optimize these anticancer and radioprotective effects. We report here
on 3,6-bis (2-oxiranylmethoxy)- 9H-xanthen-9-one, designated R71. Methods: Compound R71 was
1
synthesized beginning with 2, 4-dimethoxybenzoic acid. The structure of R71 was confirmed by H-NMR
13
and C-NMR. The antiproliferative effect of R71 in several cancer cell lines, including A549 (lung
cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer), was
measured by MTT assay. Different concentrations of R71 were added to the cell culture system for 72
hours and evaluated using the MTT assay. Apoptosis and mitochondrial stability were assayed on A549
cells using flow cytometry and with JC-1 and ROS probes. Assays were performed with and without
treatment with various doses of radiation and cisplatin. Data were analyzed with CompuSyn software.
Results: The IC50 of R71 was 19.87, 24.68, 10.3, and 13.09 μM, respectively, in the 4 cancer cell lines
tested. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R71 induced apoptosis
in A549 cells. JC-1 staining of A549 cells showed unstable mitochondrial membrane potential after R71
242 | P a g e
