Jiaying Xu

DIM (3,3′-diindolylmethane) confers protection against ionizing radiation by a unique mechanism

Significance DIM (3,3′-diindolylmethane) is a small molecule compound under investigation as a cancer preventive agent. This research addresses a potential usage of DIM as a medical countermeasure to prevent or mitigate acute radiation syndrome due to whole body exposure. In this regard, DIM can be administered safely to humans and animals by oral or subcutaneous routes. DIM may also be useful in preventing or mitigating late normal tissue damage due to partial body radiation exposure during cancer treatment. DIM works, in part, by a mechanism distinct from other radioprotectors and mitigators involving stimulation of the DNA damage response, including DNA repair, and activation of cell survival signaling through the transcription factor NF-κB. DIM (3,3′-diindolylmethane), a small molecule compound, is a proposed cancer preventive agent that can be safely administered to humans in repeated doses. We report that administration of DIM in a multidose schedule protected rodents against lethal doses of total body irradiation up to 13 Gy, whether DIM dosing was initiated before or up to 24 h after radiation. Physiologic submicromolar concentrations of DIM protected cultured cells against radiation by a unique mechanism: DIM caused rapid activation of ataxia-telangiectasia mutated (ATM), a nuclear kinase that regulates responses to DNA damage (DDR) and oxidative stress. Subsequently, multiple ATM substrates were phosphorylated, suggesting that DIM induces an ATM-dependent DDR-like response, and DIM enhanced radiation-induced ATM signaling and NF-κB activation. DIM also caused activation of ATM in rodent tissues. Activation of ATM by DIM may be due, in part, to inhibition of protein phosphatase 2A, an upstream regulator of ATM. In contrast, DIM did not protect human breast cancer xenograft tumors against radiation under the conditions tested. In tumors, ATM was constitutively phosphorylated and was not further stimulated by radiation and/or DIM. Our findings suggest that DIM is a potent radioprotector and mitigator that functions by stimulating an ATM-driven DDR-like response and NF-κB survival signaling.

The Fto Gene Regulates the Proliferation and Differentiation of Pre-Adipocytes in Vitro.

The highly regulated differentiation and proliferation of pre-adipocytes play a key role in the initiation of obesity. Fat mass and obesity associated (FTO) is a novel gene strongly associated with the risk of obesity. A deficiency of FTO may cause growth retardation in addition to fat mass and adipocyte size reduction in vivo. To investigate the potential role of Fto gene on the proliferation and differentiation of pre-adipocytes, we generated Fto-knockdown and overexpressed 3T3-L1 cells. Using numerous proliferation assays our results suggest that Fto knockdown leads to suppression of proliferation, lower mitochondrial membrane potential, less cellular ATP, and decreased and smaller intracellular lipid droplets compared with controls (p < 0.05). Western blot analysis demonstrated that Fto knockdown can significantly suppress peroxisome proliferator-activated receptor gamma (PPARγ) and glucose transporter type 4 (GLUT4) expression and inhibit Akt phosphorylation. By contrast, overexpression of Fto had the opposing effect on proliferation, mitochondrial membrane potential, ATP generation, in vitro differentiation, Akt phosphorylation, and PPARγ and GLUT4 expression. Moreover, we demonstrated that Wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, could inhibit phospho-Akt in Fto overexpressed 3T3-L1 cells. Taken together, the results suggest that Fto regulates the proliferation and differentiation of 3T3-L1 cells via multiple mechanisms, including PPARγ and PI3K/Akt signaling.

Protection effect of sanguinarine on whole-body exposure of X radiation in BALB/c mice

Erythropoietin (EPO) is a glycoprotein hormone responsible for regulating erythropoiesis. Expression of EPO and EPO receptors (EPOr) has recently been demonstrated in some neoplastic cell lines and tumours, suggesting a potential new target for therapy. In this work, EPO was labeled with iodine-125 using the lactoperoxidase method, known to prevent damage to protein during radioiodination, and labeling conditions were optimized. In vitro stability studies have shown that 125I-EPO is radiochemically stable for 20 days after radiolabeling. In vitro cell binding studies have demonstrated very low binding (<2%) of EPO to normal and neoplastic cell lines tested. As expected, the biodistribution in healthy mice exhibited comparatively high rates of fixation in the organs of the excretory system. Thyroid also proved to be a critical organ which may indicate in vivo dissociation of 125I-EPO. In mice with induced melanoma, only a residual fixation in the tumour was evident. Further studies are warranted on other tumoral cell lines to better understand the binding process and internalization into cells. Studies on EPO labeled with carbon-11 could be valuable, since there is a greater chance of preserving the biological activity of the protein using this method.

Polydatin down-regulates the phosphorylation level of Creb and induces apoptosis in human breast cancer cell

Polydatin (PD), a component isolated from Polygonum cuspidatum, has a number of biological functions. However, the antitumor activity of PD has been poorly investigated. In this study, the effect of PD on cell proliferation was evaluated by thiazolyl blue tetrazolium bromide assay. Cell cycle distribution and apoptosis were investigated by flow cytometry. The phosphorylation levels of panel of phosphor-kinases were detected by human phospho-kinase arrays. The expression of several proteins associated with cell cycle and apoptosis were analyzed by Western blot analysis. Results showed that PD effectively inhibited the growth of MDA-MB-231 and MCF-7 breast cancer cell lines. Cell cycle analysis demonstrated that PD induced S-phase cell cycle arrest. Human phosphor-kinase arrays showed that the phosphorylation level of cAMP response element-bingding proteins(Creb) was down-regulated, and the results were further confirmed by Western blot analysis. Western blot analysis showed that the expression of protein of cyclin D1 decreased in a time- and dose- dependent manner. Results suggest that PD is a potential therapeutic natural compound.

Inhibitory impacts of chemically modified tetracycline-3 and underlying mechanism in human cervical cancer cells.

Chemically modified tetracyclines (CMTs) have been rationally designed from tetracyclines. The CMTs that show the antimicrobial properties are eliminated, whereas matrix metalloproteinase inhibitory properties are retained. Interestingly, CMT-3 (COL-3, by eliminating the dimethylamino, methyl, and hydroxyl functionalities on the basic tetracycline structure), one of the CMTs, has shown strong anticancer activity. In this study, we found that CMT-3 showed dose-dependent and time-dependent cytotoxicity in HeLa and Siha cells, two human cervical cancer cell lines. HeLa cells were more sensitive to CMT-3 compared with Siha cells. The antiproliferation potential of CMT-3 was associated with the mitochondrial apoptosis pathway, increasing reactive oxygen species level, and proapoptosis protein (e.g. caspase-3) expression, but decreasing antiapoptosis protein expression (e.g. Bcl-2). N-acetylcysteine (a reactive oxygen species inhibitor) and Z-LEHD-FMK significantly reduced or blocked the apoptosis event resulting from cytotoxic effect of CMT-3. CMT-3 also induced G0/G1 phase arrest with the reduction of cell cycle regulatory protein cyclin E and the translocation of NF-&kgr;B from the cytoplasm to the nucleus. Our findings provide the important foundation for further investigation of the underlying mechanism for the anticancer activity of CMT-3 and the potential application of CMT-3 as a new therapeutic candidate for clinical cervical cancer therapy.

Novel mechanisms involving chemically modified tetracycline 3 cytotoxicity.

Chemically modified tetracycline 3 (CMT-3) is a potential anticancer drug because of its retained matrix metalloproteinases inhibitory property. In the present study, we showed that CMT-3 significantly inhibited the growth and proliferation of human hepatocellular carcinoma HepG2 cells. Novel mechanisms including increased intracellular autophagy level and high-mobility group box 1 (HMGB1) release were involved. In addition, a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS), significantly increased the cytotoxic effects of CMT-3 in HepG2 cells. Combining CMT-3 with TSN-SS led to enhanced accumulation of endogenous LC3-II, but reduced HMGB1 cytoplasmic translocation. Altogether, these findings suggest that autophagy and HMGB1 release may play important roles in the anticancer effect of CMT-3. As a novel candidate for cancer therapy, CMT-3 may be used in combination with TSN-SS, which possibly facilitates the execution of a death signal (e.g. autophagy) and prevents the survival of an inducer (e.g. HMGB1 cytoplasmic translocation), thus improving its therapeutic effect.